Method for the production of 2-butanol

ABSTRACT

A method for the production of 2-butanol by fermentation using a microbial production host is disclosed. The method employs a reduction in temperature during the fermentation process that results in a more robust tolerance of the production host to the butanol product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/915,459, filed on May 2, 2007.

FIELD OF THE INVENTION

The invention relates to a method for the production of 2-butanol byfermentation using a recombinant microbial host. Specifically, themethod employs a decrease in temperature during fermentation thatresults in more robust tolerance of the production host to the 2-butanolproduct.

BACKGROUND OF THE INVENTION

Butanol is an important industrial chemical, useful as a fuel additive,as a feedstock chemical in the plastics industry, and as a foodgradeextractant in the food and flavor industry. Each year 10 to 12 billionpounds of butanol are produced by petrochemical means and the need forthis commodity chemical will likely increase.

Methods for the chemical synthesis of 2-butanol are known, such asn-butene hydration (Ullmann's Encyclopedia of Industrial Chemistry,6^(th) edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany,Vol. 5, pp. 716-719). These processes use starting materials derivedfrom petrochemicals and are generally expensive, and are notenvironmentally friendly. The production of 2-butanol from plant-derivedraw materials would minimize greenhouse gas emissions and wouldrepresent an advance in the art.

Methods for producing 2-butanol by biotransformation of other organicchemicals are also known. For example, Stampfer et al. (WO 03/078615)describe the production of secondary alcohols, such as 2-butanol, by thereduction of ketones which is catalyzed by an alcohol dehydrogenaseenzyme obtained from Rhodococcus ruber. Similarly, Kojima et al. (EP0645453) describe a method for preparing secondary alcohols, such as2-butanol, by reduction of ketones which is catalyzed by a secondaryalcohol dehydrogenase enzyme obtained from Candida parapsilosis.Additionally, Kuehnle et al. (EP 1149918) describe a process thatproduces both 1-butanol and 2-butanol by the oxidation of hydrocarbonsby various strains of Rhodococcus ruber. The process favored 1-butanolproduction with a selectivity of 93.8%.

The production of 2-butanol by certain strains of Lactobacilli is alsoknown (Speranza et. al. J. Agric. Food Chem. (1997) 45:3476-3480). The2-butanol is produced by the transformation of meso-2,3-butanediol. Theproduction of 2-butanol from acetolactate and acetoin by theseLactobacilli strains was also demonstrated.

Recombinant microbial production hosts expressing 2-butanol biosyntheticpathways are described in co-pending and commonly owned U.S. PatentApplication Publication No. US20070259410A1. However, biologicalproduction of 2-butanol is believed to be limited by 2-butanol toxicityto the host microorganism used in the fermentation.

Some microbial strains that are tolerant to 2-butanol are known in theart (co-pending and commonly owned U.S. patent application Ser. Nos.11/743,220 and 11/761,497). However, biological methods of producing2-butanol to higher levels are required for cost effective commercialproduction.

There have been reports describing the effect of temperature on thetolerance of some microbial strains to ethanol. For example, Amartey etal. (Biotechnol. Lett. 13(9):627-632 (1991)) disclose that Bacillusstearothermophillus is less tolerant to ethanol at 70° C. than at 60° C.Herrero et al. (Appl. Environ. Microbiol. 40(3):571-577 (1980)) reportthat the optimum growth temperature of a wild-type strain of Clostridiumthermocellum decreases as the concentration of ethanol challengeincreases, whereas the optimum growth temperature of an ethanol-tolerantmutant remains constant. Brown et al. (Biotechnol. Lett. 4(4):269-274(1982)) disclose that the yeast Saccharomyces uvarum is more resistantto growth inhibition by ethanol at temperatures 5° C. and 10° C. belowits growth optimum of 35° C. However, fermentation became more resistantto ethanol inhibition with increasing temperature. Additionally, VanUden (CRC Crit. Rev. Biotechnol. 1 (3):263-273 (1984)) report thatethanol and other alkanols depress the maximum and the optimum growthtemperature for growth of Saccharomyces cerevisiae while thermal deathis enhanced. Moreover, Lewis et al. (U.S. Patent Application PublicationNo. 2004/0234649) describe methods for producing high levels of ethanolduring fermentation of plant material comprising decreasing thetemperature during saccharifying, fermenting, or simultaneouslysaccharifying and fermenting

Much less is known about the effect of temperature on the tolerance ofmicrobial strains to butanols. Harada (Hakko Kyokaishi 20:155-156(1962)) discloses that the yield of 1-butanol in acetone-butanol-ethanol(ABE) fermentation is increased from 18.4%-18.7% to 19.1%-21.2% bylowering the temperature from 30° C. to 28° C. when the growth of thebacteria reaches a maximum. Jones et al. (Microbiol. Rev. 50(4):484-524(1986)) review the role of temperature in ABE fermentation. They reportthat the solvent yields of three different solvent producing strainsremains fairly constant at 31% at 30° C. and 33° C., but decreases to 23to 25% at 37° C. Similar results were reported for Clostridiumacetobutylicum for which solvent yields decreased from 29% at 25° C. to24% at 40° C. In the latter case, the decrease in solvent yield wasattributed to a decrease in acetone production while the yield of1-butanol was unaffected. However, Carnarius (U.S. Pat. No. 2,198,104)reports that an increase in the butanol ratio is obtained in the ABEprocess by decreasing the temperature of the fermentation from 30° C. to24° C. after 16 hours. However, the effect of temperature on theproduction of 2-butanol by recombinant microbial hosts is not known inthe art.

There is a need, therefore, for a cost-effective process for theproduction of 2-butanol by fermentation that provides higher yields thanprocesses known in the art. The present invention addresses this needthrough the discovery of a method for producing 2-butanol byfermentation using a recombinant microbial host, which employs adecrease in temperature during fermentation, resulting in more robusttolerance of the production host to the 2-butanol product.

SUMMARY OF THE INVENTION

The invention provides a method for the production of 2-butanol byfermentation using a recombinant microbial host, which employs adecrease in temperature during fermentation that results in more robusttolerance of the production host to the 2-butanol product.

Accordingly, the invention provides a method for the production of2-butanol comprising:

-   -   a) providing a recombinant microbial production host which        produces 2-butanol;    -   b) seeding the production host of (a) into a fermentation medium        comprising a fermentable carbon substrate to create a        fermentation culture;    -   c) growing the production host in the fermentation culture at a        first temperature for a first period of time;    -   d) lowering the temperature of the fermentation culture to a        second temperature; and    -   e) incubating the production host at the second temperature of        step (d) for a second period of time;

whereby 2-butanol is produced.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription, FIGURE, and the accompanying sequence descriptions, whichform a part of this application.

FIG. 1 shows four different pathways for biosynthesis of 2-butanone and2-butanol.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers SEQ IDNucleic SEQ ID Description acid Protein budA, acetolactate decarboxylasefrom Klebsiella 1 2 pneumoniae ATCC 25955 alsD, acetolactatedecarboxylase from Bacillus 80 81 subtilis budA, acetolactatedecarboxylase from Klebsiella 82 83 terrigena budB, acetolactatesynthase from Klebsiella 3 4 pneumoniae ATCC 25955 alsS, acetolactatesynthase from Bacillus subtilis 76 77 budB, acetolactate synthase fromKlebsiella 78 79 terrigena budC butanediol dehydrogenase from Klebsiella5 6 pneumoniae IAM1063 butanediol dehydrogenase from Bacillus cereus 8485 butanediol dehydrogenase from Bacillus cereus 86 87 butB, butanedioldehydrogenase from Lactococcus 88 89 lactis pddA, butanediol dehydratasealpha subunit from 7 8 Klebsiella oxytoca ATCC 8724 pddB, butanedioldehydratase beta subunit from 9 10 Klebsiella oxytoca ATCC 8724 pddC,butanediol dehydratase gamma subunit from 11 12 Klebsiella oxytoca ATCC8724 pduC, B12 dependent diol dehydratase large 92 93 subunit fromSalmonella typhimurium pduD, B12 dependent diol dehydratase medium 94 95subunit from Salmonella typhimurium pduE, B12 dependent diol dehydratasesmall 96 97 subunit from Salmonella typhimurium pduC, B12 dependent dioldehydratase large 98 99 subunit from Lactobacillus collinoides pduD, B12dependent diol dehydratase medium 100 101 subunit from Lactobacilluscollinoides pduE, B12 dependent diol dehydratase small 102 103 subunitfrom Lactobacillus collinoides pddC, adenosylcobalamin-dependent diol104 105 dehydratase alpha subunit from Klebsiella pneumoniae pddD,adenosylcobalamin-dependent diol 106 107 dehydratase beta subunit fromKlebsiella pneumoniae pddD, adenosylcobalamin-dependent diol 108 109dehydratase gamma subunit from Klebsiella pneumoniae ddrA, dioldehydratase reactivating factor large 110 111 subunit from Klebsiellaoxytoca ddrB, diol dehydratase reactivating factor small 112 113 subunitfrom Klebsiella oxytoca pduG, diol dehydratase reactivating factor large114 115 subunit from Salmonella typhimurium pduH, diol dehydratasereactivating factor small 116 117 subunit from Salmonella typhimuriumpduG, diol dehydratase reactivating factor large 118 119 subunit fromLactobacillus collinoides pduH, diol dehydratase reactivating factorsmall 120 121 subunit from Lactobacillus collinoides sadH, butanoldehydrogenase from Rhodococcus 13 14 ruber 219 adhA, butanoldehydrogenase from Pyrococcus 90 91 furiosus chnA, cyclohexanoldehydrogenase from 71 72 Acinteobacter sp. yqhD, butanol dehydrogenasefrom Escherichia coli 74 75 amine: pyruvate transaminase from Vibriofluvialis 144 122 (an acetoin aminase) codon opt. amino alcohol kinasefrom Erwinia carotovora 123 124 subsp. atroseptica amino alcoholO-phosphate lyase from Erwinia 125 126 carotovora subsp. atrosepticabudC, acetoin reductase (butanediol 133 134 dehydrogenase) fromKlebsiella terrigena (now Raoultella terrigena) glycerol dehydratasealpha subunit from Klebsiella 145 146 pneumoniae glycerol dehydratasebeta subunit from Klebsiella 147 148 pneumoniae glycerol dehydratasegamma subunit from 149 150 Klebsiella pneumoniae glycerol dehydratasereactivase large subunit from 151 152 Klebsiella pneumoniae glyceroldehydratase reactivase small subunit from 153 154 Klebsiella pneumoniae

SEQ ID NOs:15-65 are the nucleotide sequences of oligonucleotide PCR,cloning, screening, and sequencing primers used in the Examples.

SEQ ID NO:66 is nucleotide sequence of the deleted region of the yqhDgene in E. coli strain MG1655 ΔyqhCD, described in Example 15.

SEQ ID NO:67 is the nucleotide sequence of a variant of the glucoseisomerase promoter 1.6GI.

SEQ ID NO:68 is the nucleotide sequence of the 1.5GI promoter.

SEQ ID NO:69 is the nucleotide sequence of the diol dehydratase operonfrom Klebsiella oxytoca.

SEQ ID NO:70 is the nucleotide sequence of the diol dehydratasereactivating factor operon from Klebsiella oxytoca.

SEQ ID NO:73 is the nucleotide sequence of pDCQ2, which is described inExample 13.

SEQ ID NOs:127-132 are the nucleotide sequences of additionaloligonucleotide PCR and cloning primers used in the Examples.

SEQ ID NO: 55 is a codon optimized coding region for the amino alcoholkinase of Erwinia carotovora subsp. atroseptica.

SEQ ID NO:156 is a codon optimized coding region for the amino alcoholO-phosphate lyase of Erwinia carotovora subsp. atroseptica.

SEQ ID NOs:157-163 are the nucleotide sequences of additionaloligonucleotide PCR and cloning primers used in the Examples.

SEQ ID NO:164 is the nucleotide sequence of an operon from Erwiniacarotovora subsp. atroseptica.

TABLE 2 Additional glycerol and diol dehydratase large, medium and smallsubunits protein ^(a)Description ^(b)subunit SEQ ID Correspondingsubunits from same organism^(c) Glycerol dehydratase alpha subunit fromClostridium L 135 pasteurianum Glycerol dehydratase beta subunit fromClostridium M 136 pasteurianum Glycerol dehydratase gamma subunit fromClostridium S 137 pasteurianum Glycerol dehydratase alpha subunit fromEscherichia L 138 blattae Glycerol dehydratase beta subunit fromEscherichia M 139 blattae Glycerol dehydratase gamma subunit fromEscherichia S 140 blattae Glycerol dehydratase alpha subunit fromCitrobacter L 141 freundii Glycerol dehydratase beta subunit fromCitrobacter M 142 freundii Glycerol dehydratase gamma subunit fromCitrobacter S 143 freundii ^(a)Description: from the Genbank annotationof the sequence and may not be correct including the glycerol or dioldesignation, or may not include subunit information. ^(b)Subunit:identified by sequence homology to the large, medium, or small subunit.of the Klebsiella oxytoca enzyme. ^(c)Subunts are listed together thatare from the same organism and have annotations as the same enzyme, orhave Genbank numbers close together indicating proximity in the genome.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the production of2-butanol using recombinant microorganisms that employs a decrease intemperature during fermentation, resulting in more robust tolerance ofthe production host to the 2-butanol product and therefore a highertiter of 2-butanol. The present invention meets a number of commercialand industrial needs. 2-Butanol is an important industrial commoditychemical with a variety of applications, where its potential as a fuelor fuel additive is particularly significant. Although only afour-carbon alcohol, butanol has an energy content similar to that ofgasoline and can be blended with any fossil fuel. Butanol is favored asa fuel or fuel additive as it yields only CO₂ and little or no SO_(X) orNO_(X) when burned in the standard internal combustion engine.Additionally butanol is less corrosive than ethanol, the most preferredfuel additive to date.

In addition to its utility as a biofuel or fuel additive, butanol hasthe potential of impacting hydrogen distribution problems in theemerging fuel cell industry. Fuel cells today are plagued by safetyconcerns associated with hydrogen transport and distribution. Butanolcan be easily reformed for its hydrogen content and can be distributedthrough existing gas stations in the purity required for either fuelcells or combustion engines in vehicles.

Finally the present invention produces 2-butanol from plant derivedcarbon sources, avoiding the negative environmental impact associatedwith standard petrochemical processes for butanol production.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, a mixture, process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but may include other elements not expresslylisted or inherent to such composition, mixture, process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances (i.e. occurrences) of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably within 5% of the reported numerical value.

The term “2-butanol biosynthetic pathway” refers to the enzyme pathwaysto produce 2-butanol from pyruvate.

The term “2-butanone biosynthetic pathway” refers to the enzyme pathwaysto produce 2-butanone from pyruvate.

The term “acetolactate synthase”, also known as “acetohydroxy acidsynthase”, refers to a polypeptide (or polypeptides) having an enzymeactivity that catalyzes the conversion of two molecules of pyruvic acidto one molecule of alpha-acetolactate. Acetolactate synthase, known asEC 2.2.1.6 [formerly 4.1.3.18] (Enzyme Nomenclature 1992, AcademicPress, San Diego) may be dependent on the cofactor thiamin pyrophosphatefor its activity. Suitable acetolactate synthase enzymes are availablefrom a number of sources, for example, Bacillus subtilis [GenBank Nos:AAA22222 NCBI (National Center for Biotechnology Information) amino acidsequence (SEQ ID NO:77), L04470 NCBI nucleotide sequence (SEQ IDNO:76)], Klebsiella terrigena [GenBank Nos: AAA25055 (SEQ ID NO:79),L04507 (SEQ ID NO:78)], and Klebsiella pneumoniae [GenBank Nos: AAA25079(SEQ ID NO:4), M73842 (SEQ ID NO:3)].

The term “acetolactate decarboxylase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofalpha-acetolactate to acetoin. Acetolactate decarboxylases are known asEC 4.1.1.5 and are available, for example, from Bacillus subtilis[GenBank Nos: AAA22223 (SEQ ID NO:81), L04470 (SEQ ID NO:80)],Klebsiella terrigena [GenBank Nos: AAA25054 (SEQ ID NO:83), L04507 (SEQID NO:82)] and Klebsiella pneumoniae [GenBank Nos: AAU43774 (SEQ IDNO:2), AY722056 (SEQ ID NO:1)].

The term “acetoin aminase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of acetoin to3-amino-2-butanol. Acetoin aminase may utilize the cofactor pyridoxal5′-phosphate or NADH (reduced nicotinamide adenine dinucleotide) orNADPH (reduced nicotinamide adenine dinucleotide phosphate). Theresulting product may have (R) or (S) stereochemistry at the 3-position.The pyridoxal phosphate-dependent enzyme may use an amino acid such asalanine or glutamate as the amino donor. The NADH- and NADPH-dependentenzymes may use ammonia as a second substrate. A suitable example of anNADH-dependent acetoin aminase, also known as amino alcoholdehydrogenase, is described by Ito et al. (U.S. Pat. No. 6,432,688). Anexample of a pyridoxal-dependent acetoin aminase is the amine:pyruvateaminotransferase (also called amine:pyruvate transaminase) described byShin and Kim (J. Org. Chem. 67:2848-2853 (2002)).

The term “butanol dehydrogenase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes theinterconversion of 2-butanone and 2-butanol. Butanol dehydrogenases area subset of a broad family of alcohol dehydrogenases. Butanoldehydrogenase may be NAD- or NADP-dependent. The NAD-dependent enzymesare known as EC 1.1.1.1 and are available, for example, from Rhodococcusruber [GenBank Nos: CAD36475 (SEQ ID NO:14), AJ491307 (SEQ ID NO:13)].The NADP-dependent enzymes are known as EC 1.1.1.2 and are available,for example, from Pyrococcus furiosus [GenBank Nos: AAC25556 (SEQ IDNO:91), AF013169 (SEQ ID NO:90)]. Additionally, a butanol dehydrogenaseis available from Escherichia coli [GenBank Nos:NP_(—)417-484 (SEQ IDNO:75), NC_(—)000913 (SEQ ID NO:74)] and a cyclohexanol dehydrogenasewith activity towards 2-butanol is available from Acinetobacter sp.[GenBank Nos: AAG10026 (SEQ ID NO:72), AF282240 (SEQ ID NO:71)].

The term “acetoin kinase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of acetoin tophosphoacetoin. Acetoin kinase may utilize ATP (adenosine triphosphate)or phosphoenolpyruvate as the phosphate donor in the reaction. Althoughthere are no reports of enzymes catalyzing this reaction on acetoin,there are enzymes that catalyze the analogous reaction on the similarsubstrate dihydroxyacetone, for example, enzymes known as EC 2.7.1.29(Garcia-Alles et al. (2004) Biochemistry 43:13037-13046).

The term “acetoin phosphate aminase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofacetoin phosphate (also called phosphoacetoin) to 3-amino-2-butanolO-phosphate. Acetoin phosphate aminase may use the cofactor pyridoxal5′-phosphate, NADH or NADPH. The resulting product may have (R) or (S)stereochemistry at the 3-position. The pyridoxal phosphate-dependentenzyme may use an amino acid such as alanine or glutamate. The NADH- andNADPH-dependent enzymes may use ammonia as a second substrate. Althoughthere are no reports of enzymes catalyzing this reaction on acetoinphosphate, there is a pyridoxal phosphate-dependent enzyme that isproposed to carry out the analogous reaction on the similar substrateserinol phosphate (Yasuta et al. (2001) Appl. Environ. Microbiol.67:4999-5009).

The term “aminobutanol phosphate phospho-lyase”, also called “aminoalcohol O-phosphate lyase”, refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of3-amino-2-butanol O-phosphate to 2-butanone. Aminobutanol phosphatephospho-lyase may utilize the cofactor pyridoxal 5′-phosphate. There arereports of enzymes that catalyze the analogous reaction on the similarsubstrate 1-amino-2-propanol phosphate (Jones et al. (1973) Biochem J.134:167-182). Disclosed in co-owned and co-pending US Patent ApplicationPublication No. 20070259410A1 is an aminobutanol phosphate phospho-lyase(SEQ ID NO: 126) from the organism Erwinia carotovora, with demonstratedaminobutanol phosphate phospho-lyase activity.

The term “aminobutanol kinase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of3-amino-2-butanol to 3-amino-2-butanol O-phosphate. Aminobutanol kinasemay utilize ATP as the phosphate donor. There are reports of enzymesthat catalyze the analogous reaction on the similar substratesethanolamine and 1-amino-2-propanol (Jones et al., supra). Disclosed inco-owned and co-pending US Patent Application Publication No.20070259410A1 is an amino alcohol kinase of Erwinia carotovora subsp.atroseptica (SEQ ID NO:124).

The term “butanediol dehydrogenase” also known as “acetoin reductase”refers to a polypeptide (or polypeptides) having an enzyme activity thatcatalyzes the conversion of acetoin to 2,3-butanediol. Butanedioldehydrogenases are a subset of the broad family of alcoholdehydrogenases. Butanediol dehydrogenase enzymes may have specificityfor production of (R)- or (S)-stereochemistry in the alcohol product.(S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and areavailable, for example, from Klebsiella pneumoniae (GenBank Nos:BBA13085 (SEQ ID NO:6), D86412 (SEQ ID NO:5)). (R)-specific butanedioldehydrogenases are known as EC 1.1.1.4 and are available, for example,from Bacillus cereus [GenBank Nos. NP_(—)830481 (SEQ ID NO:85),NC_(—)004722 (SEQ ID NO:84); AAP07682 (SEQ ID NO:87), AE017000 (SEQ IDNO:86)], and Lactococcus lactis [GenBank Nos. AAK04995 (SEQ ID NO:89),AE006323 (SEQ ID NO:88)].

The term “butanediol dehydratase”, also known as “diol dehydratase” or“propanediol dehydratase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of2,3-butanediol to 2-butanone. Butanediol dehydratase may utilize thecofactor adenosyl cobalamin (vitamin B12). Adenosyl cobalamin-dependentenzymes are known as EC 4.2.1.28 and are available, for example, fromKlebsiella oxytoca [GenBank Nos: BAA08099 (alpha subunit) (SEQ ID NO:8),D45071 (SEQ ID NO:7); BAA08100 (beta subunit) (SEQ ID NO:10), D45071(SEQ ID NO:9); and BBA08101 (gamma subunit) (SEQ ID NO:12), D45071 (SEQID NO:11) (Note all three subunits are required for activity)], andKlebsiella pneumoniae [GenBank Nos: AAC98384 (alpha subunit) (SEQ IDNO:105), AF102064 (SEQ ID NO:104); GenBank Nos: AAC98385 (beta subunit)(SEQ ID NO:107), AF102064 (SEQ ID NO:106), GenBank Nos: AAC98386 (gammasubunit) SEQ ID NO:109), AF102064 (SEQ ID NO:108)]. Other suitable dioldehydratases include, but are not limited to, B12-dependent dioldehydratases available from Salmonella typhimurium [GenBank Nos:AAB84102 (large subunit) (SEQ ID NO:93), AF026270 (SEQ ID NO:92);GenBank Nos: AAB84103 (medium subunit) (SEQ ID NO:95), AF026270 (SEQ IDNO:94); GenBank Nos: AAB84104 (small subunit) (SEQ ID NO:97), AF026270(SEQ ID NO:96)]; and Lactobacillus collinoides [GenBank Nos: CAC82541(large subunit) (SEQ ID NO:99), AJ297723 (SEQ ID NO:98); GenBank Nos:CAC82542 (medium subunit) (SEQ ID NO:101); AJ297723 (SEQ ID NO:100);GenBank Nos: CAD01091 (small subunit) (SEQ ID NO:103), AJ297723 (SEQ IDNO:102)]; and enzymes from Lactobacillus brevis (particularly strainsCNRZ 734 and CNRZ 735, Speranza et al., supra), and nucleotide sequencesthat encode the corresponding enzymes. Methods of diol dehydratase geneisolation are well known in the art (e.g., U.S. Pat. No. 5,686,276).

The term “glycerol dehydratase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofglycerol to 3-hydroxypropionaldehyde. Adenosyl cobalamin-dependentglycerol dehydratases are known as EC 4.2.1.30. The glyceroldehydratases of EC 4.2.1.30 are similar to the diol dehydratases insequence and in having three subunits. The glycerol dehydratases canalso be used to convert 2,3-butanediol to 2-butanone. Some examples ofglycerol dehydratases of EC 4.2.1.30 include those from Klebsiellapneumoniae (alpha subunit, SEQ ID NO:145, coding region and SEQ IDNO:146, protein; beta subunit, SEQ ID NO:147, coding region and SEQ IDNO:148, protein; and gamma subunit SEQ ID NO:149, coding region and SEQID NO:150, protein); from Clostridium pasteurianum [GenBank Nos: 3360389(alpha subunit, SEQ ID NO:135), 3360390 (beta subunit, SEQ ID NO:136),and 3360391 (gamma subunit, SEQ ID NO:137)]; from Escherichia blattae[GenBank Nos: 60099613 (alpha subunit, SEQ ID NO:138), 57340191 (betasubunit, SEQ ID NO:139), and 57340192 (gamma subunit, SEQ ID NO:140)];and from Citrobacter freundii [GenBank Nos: 1169287 (alpha subunit, SEQID NO:141), 1229154 (beta subunit, SEQ ID NO:142), and 1229155 (gammasubunit, SEQ ID NO:143)]. Note that all three subunits are required foractivity. Additional glycerol dehydratases are listed in Table 2.

Diol and glycerol dehydratases may undergo suicide inactivation duringcatalysis. A reactivating factor protein, also referred to herein as“reactivase”, can be used to reactivate the inactive enzymes (Mori etal., J. Biol. Chem. 272:32034 (1997)). Preferably, the reactivatingfactor is obtained from the same source as the diol or glyceroldehydratase used. For example, suitable diol dehydratase reactivatingfactors are available from Klebsiella oxytoca [GenBank Nos: AAC15871(large subunit) (SEQ ID NO:11), AF017781 (SEQ ID NO:10); GenBank Nos:AAC15872 (small subunit) (SEQ ID NO:113), AF017781 (SEQ ID NO:112)];Salmonella typhimurium [GenBank Nos: AAB84105 (large subunit) (SEQ IDNO:115), AF026270 (SEQ ID NO:114), GenBank Nos: AAD39008 (small subunit)(SEQ ID NO:117), AF026270 (SEQ ID NO:116)]; and Lactobacilluscollinoides [GenBank Nos: CAD01092 (large subunit) (SEQ ID NO:119),AJ297723 (SEQ ID NO:118); GenBank Nos: CAD01093 (small subunit) (SEQ IDNO:121), AJ297723 (SEQ ID NO:120)]. Both the large and small subunitsare required for activity. For example, suitable glycerol dehydratasereactivating factors are available from Klebsiella pneumoniae (largesubunit, SEQ ID NO:151, coding region and SEQ ID NO:152, protein; andsmall subunit, SEQ ID NO:153, coding region and SEQ ID NO:154, protein).

The term “a facultative anaerobe” refers to a microorganism that cangrow in both aerobic and anaerobic environments.

The term “carbon substrate” or “fermentable carbon substrate” refers toa carbon source capable of being metabolized by host organisms disclosedherein and particularly carbon sources selected from the groupconsisting of monosaccharides, oligosaccharides, polysaccharides, andone-carbon substrates or mixtures thereof.

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

As used herein, an “isolated nucleic acid fragment” or “isolated nucleicacid molecule” or “genetic construct” will be used interchangeably andwill mean a polymer of RNA or DNA that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAmay be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid fragment can anneal to theother nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washeswith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Altschul, S. F., et al.,J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten ormore contiguous amino acids or thirty or more nucleotides is necessaryin order to putatively identify a polypeptide or nucleic acid sequenceas homologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches the complete amino acidand nucleotide sequence encoding particular proteins. The skilledartisan, having the benefit of the sequences as reported herein, may nowuse all or a substantial portion of the disclosed sequences for purposesknown to those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

The terms “homology” and “homologous” are used interchangeably herein.They refer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that homologous nucleic acidsequences encompassed by this invention are also defined by theirability to hybridize, under moderately stringent conditions (e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesis performed using the “Clustal method of alignment” which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMegAlign™ program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values correspondto GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters forpairwise alignments and calculation of percent identity of proteinsequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences using the Clustal V program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram. Additionally the “Clustal W method of alignment” is availableand corresponds to the alignment method labeled Clustal W (described byHiggins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al.,Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to: 24%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%, or any integer percentage from 24% to 100% may beuseful in describing the present invention, such as 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%. Suitable nucleic acid fragments not only have the above homologiesbut typically encode a polypeptide having at least 50 amino acids,preferably at least 100 amino acids, more preferably at least 150 aminoacids, still more preferably at least 200 amino acids, and mostpreferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

As used herein the term “coding sequence” or “CDS” refers to a DNAsequence that codes for a specific amino acid sequence. “Suitableregulatory sequences” refer to nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment disclosed herein. Expression may also refer totranslation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “plasmid” and “vector” refer to an extra chromosomal elementoften carrying genes which are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA molecules.Such elements may be autonomously replicating sequences, genomeintegrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing apromoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell. “Transformationvector” refers to a specific vector containing a foreign gene and havingelements in addition to the foreign gene that facilitates transformationof a particular host cell.

As used herein the term “codon degeneracy” refers to the nature in thegenetic code permitting variation of the nucleotide sequence withoutaffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA.

The term “fermentation product medium” refers to a medium in whichfermentation has occurred such that product is present in the medium.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

2-Butanol Biosynthetic Pathways

Carbohydrate utilizing microorganisms employ the Embden-Meyerhof-Parnas(EMP) pathway, the Entner-Doudoroff pathway and the pentose phosphatecycle as the central, metabolic routes to provide energy and cellularprecursors for growth and maintenance. These pathways have in common theintermediate glyceraldehyde 3-phosphate, and, ultimately, pyruvate isformed directly or in combination with the EMP pathway. The combinedreactions of sugar conversion to pyruvate produce energy (e.g. adenosine5′-triphosphate, ATP) and reducing equivalents (e.g. reducednicotinamide adenine dinucleotide, NADH, and reduced nicotinamideadenine dinucleotide phosphate, NADPH). NADH and NADPH must be recycledto their oxidized forms (NAD⁺ and NADP⁺, respectively). In the presenceof inorganic electron acceptors (e.g. O₂, NO₃ ⁻ and SO₄ ²⁻), thereducing equivalents may be used to augment the energy pool;alternatively, a reduced carbon by-product may be formed.

As described in co-owned and co-pending US Patent ApplicationPublication Nos. 20070259410A1 and 20070292927A1,2-butanol can beproduced from carbohydrate sources in recombinant microorganismscomprising a complete 2-butanol biosynthetic pathway. Four biosyntheticpathways including all steps starting with pyruvate for production of2-butanol are shown in FIG. 1. The letters and roman numerals citedbelow correspond to the letters and roman numerals in FIG. 1, which areused to depict the conversion steps and products, respectively. Asdescribed below, 2-butanone is an intermediate in all of these 2-butanolbiosynthetic pathways.

All of the pathways begin with the initial reaction of two pyruvatemolecules to yield alpha-acetolactate (I), shown as the substrate toproduct conversion (a) in FIG. 1. From alpha-acetolactate, there are 4possible pathways to 2-butanone (V), referred to herein as 2-butanonebiosynthetic pathways:

-   -   Pathway 1) I->II->III->IV->V (substrate to product conversions        b, c, d, e)        -   2) I->II->VII->IV->V (substrate to product conversions b, g,            h, e)        -   3) I->II->VIII->V (substrate to product conversions b, i, j)        -   4) I->IX->X->V (substrate to product conversions k, l, m)            The 2-butanol biosynthetic pathways conclude with the            conversion of 2-butanone (V) to 2-butanol (VI). A detailed            discussion of the substrate to product conversions in each            pathway is given below.            Pathway 1:

(a) pyruvate to alpha-acetolactate

The initial step in pathway 1 is the conversion of two molecules ofpyruvate to one molecule of alpha-acetolactate (compound I in FIG. 1)and one molecule of carbon dioxide catalyzed by a thiaminpyrophosphate-dependent enzyme. Enzymes catalyzing this substrate toproduct conversion (generally called either acetolactate synthase oracetohydroxy acid synthase; EC 2.2.1.6 [switched from 4.1.3.18 in 2002])are well-known, and they participate in the biosynthetic pathway for theproteinogenic amino acids leucine and valine, as well as in the pathwayfor fermentative production of 2,3-butanediol and acetoin of a number oforganisms.

The skilled person will appreciate that polypeptides having acetolactatesynthase activity isolated from a variety of sources will be useful inpathway 1 independent of sequence homology. Some examples of suitableacetolactate synthase enzymes are available from a number of sources,for example, Bacillus subtilis [GenBank Nos: AAA22222 NCBI (NationalCenter for Biotechnology Information) amino acid sequence (SEQ IDNO:77), L04470 NCBI nucleotide sequence (SEQ ID NO:76)], Klebsiellaterrigena [GenBank Nos: AAA25055 (SEQ ID NO:79), L04507 (SEQ ID NO:78)],and Klebsiella pneumoniae [GenBank Nos: AAA25079 (SEQ ID NO:4), M73842(SEQ ID NO:3)]. Preferred acetolactate synthase enzymes are those thathave at least 80%-85% identity to SEQ ID NO's 4, 77, and 79, where atleast 85%-90% identity is more preferred and where at least 95% identitybased on the Clustal W method of alignment using the default parametersof GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series ofprotein weight matrix, is most preferred.

(b) alpha-acetolactate to acetoin

Alpha-acetolactate (I) is converted to acetoin (II) by the action of anenzyme such as acetolactate decarboxylase (EC 4.1.1.5). Likeacetolactate synthase, this enzyme is thiamin pyrophosphate-dependentand is also involved in the production of 2,3-butanediol and acetoin bya number of organisms. The enzymes from different sources vary quitewidely in size (25-50 kilodaltons), oligomerization (dimer-hexamer),localization (intracellular of extracellular), and allosteric regulation(for example, activation by branched-chain amino acids). Anintracellular location is preferable to extracellular, but othervariations are generally acceptable.

The skilled person will appreciate that polypeptides having acetolactatedecarboxylase activity isolated from a variety of sources will be usefulin pathway 1 independent of sequence homology. Some example of suitableacetolactate decarboxylase enzymes are available from a number ofsources, for example, Bacillus subtilis [GenBank Nos: AAA22223 (SEQ IDNO:81), L04470 (SEQ ID NO:80)], Klebsiella terrigena [GenBank Nos:AAA25054 (SEQ ID NO:83), L04507 (SEQ ID NO:82)] and Klebsiellapneumoniae [GenBank Nos: AAU43774 (SEQ ID NO:2), AY722056 (SEQ IDNO:1)].

Preferred acetolactate decarboxylase enzymes are those that have atleast 80%-85% identity to SEQ ID NO's 2, 81 and 83, where at least85%-90% identity is more preferred and where at least 95% identity basedon the Clustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix, is most preferred.

(c) acetoin to 3-amino-2-butanol

There are two known types of biochemical reactions that could effect thesubstrate to product conversion of acetoin (II) to 3-amino-2-butanol(III), specifically, pyridoxal phosphate-dependent transaminationutilizing an accessory amino donor and direct reductive amination withammonia. In the latter case, the reducing equivalents are supplied inthe form of a reduced nicotinamide cofactor (either NADH or NADPH). Anexample of an NADH-dependent enzyme catalyzing this reaction withacetoin as a substrate is reported by Ito et al. (U.S. Pat. No.6,432,688). Any stereospecificity of this enzyme has not been assessed.An example of a pyridoxal phosphate-dependent transaminase thatcatalyzes the conversion of acetoin to 3-amino-2-butanol has beenreported by Shin and Kim (supra). This enzyme was shown in co-owned andco-pending US Patent Application Publication No. 20070259410A1 toconvert both the (R) isomer of acetoin to the (2R,3S) isomer of3-amino-2-butanol and the (S) isomer of acetoin to the (2S,3S) isomer of3-amino-2-butanol. Either type of enzyme (i.e., transaminase orreductive aminase) is considered to be an acetoin aminase and may beutilized in the production of 2-butanol. Other enzymes in this group mayhave different stereospecificities.

The skilled person will appreciate that polypeptides having acetoinaminase activity isolated from a variety of sources will be useful inthe present invention independent of sequence homology. One example of aprotein having this activity is described in co-owned and co-pending USPatent Application Publication No. 20070259410A1 (SEQ ID NO:122).Accordingly preferred acetoin aminase enzymes are those that have atleast 80%-85% identity to SEQ ID NO:122, where at least 85%-90% identityis more preferred and where at least 95% identity based on the Clustal Wmethod of alignment using the default parameters of GAP PENALTY=10, GAPLENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix, ismost preferred.

(d) 3-amino-2-butanol to 3-amino-2-butanol O-phosphate

There are no enzymes known in the art that catalyze the substrate toproduct conversion of 3-amino-2-butanol (III) to 3-amino-2-butanolphosphate (IV). However, a few Pseudomonas and Erwinia species have beenshown to express an ATP-dependent ethanolamine kinase (EC 2.7.1.82)which allows them to utilize ethanolamine or 1-amino-2-propanol as anitrogen source (Jones et al. (1973) Biochem. J. 134:167-182). It islikely that this enzyme also has activity towards 3-amino-2-butanol orcould be engineered to do so, thereby providing an aminobutanol kinase.Disclosed in co-owned and co-pending US Patent Application PublicationNo. 20070259410A1 is a gene of Erwinia carotovora subsp. atroseptica(SEQ ID NO:123) that encodes a protein (SEQ ID NO:24) identified as anamino alcohol kinase. This enzyme may be used to convert3-amino-2-butanol to 3-amino-2-butanol O-phosphate.

The skilled person will appreciate that polypeptides having aminobutanolkinase activity isolated from a variety of sources will be useful in thepresent invention independent of sequence homology. One example of thisactivity is described in co-owned and co-pending US Patent ApplicationPublication No. 20070259410A1 (SEQ ID NO:124). Accordingly preferredaminobutanol kinase enzymes are those that have at least 80%-85%identity to SEQ ID NO:124, where at least 85%-90% identity is morepreferred and where at least 95% identity based on the Clustal W methodof alignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix, is mostpreferred.

(e) 3-amino-2-butanol phosphate to 2-butanone

Although there are no enzymes reported to catalyze the substrate toproduct conversion of 3-amino-2-butanol phosphate (IV) to 2-butanone(V), the substrate is very similar to those utilized by the pyridoxalphosphate-dependent phosphoethanolamine phospho-lyase enzyme, which hasbeen found in a small number of Pseudomonas and Erwinia species. Theseenzymes have activity towards phosphoethanolamine and both enantiomersof 2-phospho-1-aminopropane (Jones et al. (1973) Biochem. J.134:167-182), and may also have activity towards 3-amino-2-butanolO-phosphate. Applicants have identified a gene of Erwinia carotovorasubsp. atroseptica (SEQ ID NO:125) that encodes a protein (SEQ IDNO:126) with homology to class III aminotransferases was identified. Itwas shown to have activity on both aminopropanol phosphate andaminobutanol phosphate substrates. The enzyme was able to catalyze theconversion of a mixture of (R)-3-amino-(S)-2-butanol and(S)-3-amino-(R)-2-butanol O-phosphate, and a mixture of(R)-3-amino-(R)-2-butanol and (S)-3-amino-(S)-2-butanol O-phosphate to2-butanone. The enzyme was also able to catalyze the conversion of both(R) and (S)-2-amino-1-propanol phosphate to propanone, with a preferencefor (S)-2-amino-1-propanol phosphate. The highest activity was with theproposed natural substrate DL-1-amino-2-propanol phosphate, which wasconverted to propionaldehyde.

The skilled person will appreciate that polypeptides having aminobutanolphosphate phospho-lyase activity isolated from a variety of sources willbe useful in the present invention independent of sequence homology. Oneexample of a suitable aminobutanol phosphate phospho-lyase enzyme isdescribed in co-owned and co-pending US Patent Application PublicationNo. 20070259410A1 (SEQ ID NO: 126). Accordingly preferred aminobutanolphosphate phospho-lyase enzymes are those that have at least 80%-85%identity to SEQ ID NO's 126, where at least 85%-90% identity is morepreferred and where at least 95% identity based on the Clustal W methodof alignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix, is mostpreferred.

(f) 2-butanone to 2-butanol

The final step in all pathways to produce 2-butanol from pyruvic acid isthe reduction of 2-butanone (V) to 2-butanol (VI). This substrate toproduct conversion is catalyzed by some members of the broad class ofalcohol dehydrogenases (types utilizing either NADH or NADPH as a sourceof hydride, depending on the enzyme) that may be called butanoldehydrogenases. Enzymes of each type that catalyze the reduction of2-butanone are well known, as described above in the definition forbutanol dehydrogenase.

The skilled person will appreciate that polypeptides having butanoldehydrogenase activity isolated from a variety of sources will be usefulin the present invention independent of sequence homology. Some exampleof suitable butanol dehydrogenase enzymes are available from a number ofsources, for example, Rhodococcus ruber [GenBank Nos: CAD36475 (SEQ IDNO:14), AJ491307 (SEQ ID NO:13)]. The NADP-dependent enzymes are knownas EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus[GenBank Nos: AAC25556 (SEQ ID NO:91), AF013169 (SEQ ID NO:90)].Additionally, a butanol dehydrogenase is available from Escherichia coli[GenBank Nos:NP_(—)417-484 (SEQ ID NO:75), NC_(—)000913 (SEQ ID NO:74)]and a cyclohexanol dehydrogenase is available from Acinetobacter sp.[GenBank Nos: AAG10026 (SEQ ID NO:72), AF282240 (SEQ ID NO:71)].Preferred butanol dehydrogenase enzymes are those that have at least80%-85% identity to SEQ ID NO's 14, 91, 75, and 72, where at least85%-90% identity is more preferred and where at least 95% identity basedon the Clustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix, is most preferred.

Pathway 2:

(a) pyruvate to alpha-acetolactate

This substrate to product conversion is the same as described above forPathway 1.

(b) alpha-acetolactate to acetoin

This substrate to product conversion is the same as described above forPathway 1.

(g) acetoin to phosphoacetoin

Although enzymes that catalyze the substrate to product conversion ofacetoin (II) to phosphoacetoin (VII) have not been described, thestructure of the substrate acetoin is very similar to that ofdihydroxyacetone, and therefore acetoin may be an acceptable substratefor dihydroxyacetone kinase (EC 2.7.1.29), an enzyme which catalyzesphosphorylation of dihydroxyacetone. Protein engineering techniques forthe alteration of substrate specificity of enzymes are well known(Antikainen and Martin (2005) Bioorg. Med. Chem. 13:2701-2716) and maybe used to generate an enzyme with the required specificity. In thisconversion, the phosphate moiety may be supplied by any high energybiological phosphate donor, with the common substrates beingphosphoenolpyruvate (as in the E. coli dihydroxyacetone kinase) and ATP(as in the Citrobacter freundii dihydroxyacetone kinase) (Garcia-Alleset al. (2004) Biochemistry 43:13037-13045).

(h) phosphoacetoin to 3-amino-2-butanol O-phosphate

Although enzymes that catalyze the substrate to product conversion ofphosphoacetoin (VII) to 3-amino-2-butanol O-phosphate (IV) have not beendescribed, the structure of the substrate is very similar to that ofdihydroxyacetone phosphate, a substrate for the proposed serinolphosphate aminotransferase encoded by the 5′ portion of the rtxA gene insome species of Bradyrhizobium (Yasuta et al., supra). Thus a serinolphosphate aminotransferase may be functional in this step.

(e) 3-amino-2-butanol O-phosphate to 2-butanone

This substrate to product conversion is the same as described above forPathway 1.

(f) 2-butanone to 2-butanol

This substrate to product conversion is the same as described above forPathway 1.

Pathway 3:

(a) pyruvate to alpha-acetolactate

This substrate to product conversion is the same as described above forPathway 1.

(b) alpha-acetolactate to acetoin

This substrate to product conversion is the same as described above forPathway 1.

(i) acetoin to 2,3-butanediol

The substrate to product conversion of acetoin (II) to 2,3-butanediol(VII) may be catalyzed by a butanediol dehydrogenase that may eitherutilize NADH or NADPH as the source of reducing equivalents whencarrying out reductions. Enzymes with activity towards acetoinparticipate in the pathway for production of 2,3-butanediol in organismsthat produce that compound. The reported enzymes (e.g., BudC fromKlebsiella pneumoniae (Ui et al. (2004) Letters in Applied Microbiology39:533-537) generally utilize NADH. Either cofactor is acceptable foruse in the production of 2-butanol by this pathway.

(j) 2,3-butanediol to 2-butanone

The substrate to product conversion of 2,3-butanediol (VIII) to2-butanone (V) may be catalyzed by diol dehydratase enzymes (EC4.2.1.28) and glycerol dehydratase enzymes (EC 4.2.1.30). The bestcharacterized diol dehydratase is the coenzyme B12-dependent Klebsiellaoxytoca enzyme, but similar enzymes are found in many enteric bacteria.The K. oxytoca enzyme has been shown to accept meso-2,3-butanediol as asubstrate (Bachovchin et al. (1977) Biochemistry 16:1082-1092),producing the desired product 2-butanone. Applicants describe aKlebsiella pneumoniae glycerol dehydratase able to convertmeso-2,3-butanediol to 2-butanone. The three subunit of the Klebsiellapneumoniae glycerol dehydratase (alpha: SEQ ID NO:145 (coding region)and 146 (protein); beta: SEQ ID NO: 147 (coding region) and 148(protein); and gamma: SEQ ID NO: 149 (coding region) and 150 (protein))were expressed in conjunction with the two subunits of the Klebsiellapneumoniae glycerol dehydratase reactivase (large subunit, SEQ ID NO:151 (coding region) and 152 (protein); and small subunit, SEQ ID NO: 153(coding region) and 154 (protein)) to provide activity.

There are also reports in the literature of a B12-independent dioldehydratase from Clostridium glycolicum (Hartmanis et al. (1986) Arch.Biochem. Biophys. 245:144-152). This enzyme has activity towards2,3-butanediol, although this activity is less than 1% of the activitytowards ethanediol, but the enzyme may be engineered to improve thatactivity. A better-characterized B12-independent dehydratase is theglycerol dehydratase from Clostridium butyricum (O'Brien et al. (2004)Biochemistry 43:4635-4645), which has high activity towards1,2-propanediol as well as glycerol. This enzyme usesS-adenosylmethionine as a source of adenosyl radical. There are noreports of activity towards 2,3-butanediol, but such activity, if notalready present, may possibly be engineered.

The skilled person will appreciate that polypeptides having butanedioldehydrogenase activity isolated from a variety of sources will be usefulin the present invention independent of sequence homology. As notedabove a variety of diol and glycerol dehydratases have been described inthe literature and will be suitable for use in the present invention.Accordingly, in one aspect of the invention preferred diol and glyceroldehydratase enzymes are those that have at least 80%-85% identity toenzymes having the large, medium and small subunits, respectively of thesequences listed below:

a) SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12;

b) SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:97;

c) SEQ ID NO:99, SEQ ID NO:101, and SEQ ID NO:103;

d) SEQ ID NO:105, SEQ ID NO:107, and SEQ ID NO:109;

e) SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137;

f) SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140;

g) SEQ ID NO:146, SEQ ID NO:148, and SEQ ID NO:150;

h) SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143; and

i) SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166.

where at least 85%-90% identity is more preferred and where at least 95%identity based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix, is most preferred.

Similarly preferred diol and glycerol dehydratase enzymes are those thathave at least 80%-85% identity to enzymes having the large, medium andsmall subunits, respectively of the sequences listed below: Largesubunit: SEQ ID NOs: 8, 99, 105, 135, 138, 141, 146, and 164; Mediumsubunit: SEQ ID NOs: 10, 101, 107, 136, 139, 142, 148, and 165; Smallsubunit: SEQ ID NOs:12, 103, 109, 137, 140, 143, 150, and 166; where atleast 85%-90% identity is more preferred and where at least 95% identitybased on the Clustal W method of alignment using the default parametersof GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series ofprotein weight matrix, is most preferred.

(f) 2-butanone to 2-butanol

This substrate to product conversion is the same as described above forPathway 1.

Pathway 4:

(a) pyruvate to alpha-acetolactate

This substrate to product conversion is the same as described above forPathway 1.

(k) alpha-acetolactate to 2,3-dihydroxy-2-methylbutanoic acid

The substrate to product conversion of acetolactate (I) to2,3-dihydroxy-2-methylbutanoic acid (IX) is not known in the art.However, the product of this conversion has been reported as a componentof fermentation broths (Ziadi et al. (1973) Comptes Rendus des Seancesde l'Academie des Sciences, Serie D: Sciences Naturelles 276:965-8), butthe mechanism of formation is unknown. The likely mechanism of formationis reduction of acetolactate with NADH or NADPH as the electron donor.To utilize this pathway for production of 2-butanol, an enzymecatalyzing this reaction needs to be identified or engineered. However,the precedent for enzymatic reduction of ketones to alcohols is wellestablished.

(l) 2,3-dihydroxy-2-methylbutanoic acid to2-hydroxy-2-methyl-3-phosphobutanoic acid

There are no enzymes known that catalyze the substrate to productconversion of 2,3-dihydroxy-2-methylbutanoic acid (IX) to2-hydroxy-2-methyl-3-phosphobutanoic acid (X). However, there are alarge number of kinases in Nature that possess varying specificity. Itis therefore likely that an enzyme could be isolated or engineered withthis activity.

(m) 2-hydroxy-2-methyl-3-phosphobutanoic acid to 2-butanone

There are no known enzymes that catalyze the substrate to productconversion of 2-hydroxy-2-methyl-3-phosphobutanoic acid (X) to2-butanone (V). The combination of this reaction with the previous oneis very similar to the multi-step reaction catalyzed bymevalonate-5-pyrophosphate (M5PP) decarboxylase, which consists ofinitial phosphorylation of M5PP to 3-phosphomevalonate-5-PP, followed bydecarboxylation-dependent elimination of phosphate (Alvear et al. (1982)Biochemistry 21:4646-4650).

(f) 2-butanone to 2-butanol

This substrate to product conversion is the same as described above forPathway 1.

Thus, in providing multiple recombinant pathways from pyruvate to2-butanol, there exists a number of choices to fulfill the individualconversion steps, and the person of skill in the art will be able toutilize publicly available sequences and sequences disclosed herein toconstruct the relevant pathways. A listing of a representative number ofgenes known in the art and useful in the construction of 2-butanolbiosynthetic pathways is given above in Tables 1 and 2.

Microbial Hosts for 2-Butanol and 2-Butanone Production

Microbial hosts for 2-butanol or 2-butanone production may be selectedfrom bacteria, cyanobacteria, filamentous fungi and yeasts. Themicrobial host used for 2-butanol or 2-butanone production should betolerant to the product produced, so that the yield is not limited bytoxicity of the product to the host. The selection of a microbial hostfor 2-butanol production is described in detail below.

Microbes that are metabolically active at high titer levels of 2-butanolare not well known in the art. Although butanol-tolerant mutants havebeen isolated from solventogenic Clostridia, little information isavailable concerning the butanol tolerance of other potentially usefulbacterial strains. Most of the studies on the comparison of alcoholtolerance in bacteria suggest that butanol is more toxic than ethanol(de Cavalho et al., Microsc. Res. Tech. 64:215-22 (2004) and Kabelitz etal., FEMS Microbiol. Lett. 220:223-227 (2003)). Tomas et al. (J.Bacteriol. 186:2006-2018 (2004)) report that the yield of 1-butanolduring fermentation in Clostridium acetobutylicum may be limited bybutanol toxicity. The primary effect of 1-butanol on Clostridiumacetobutylicum is disruption of membrane functions (Hermann et al.,Appl. Environ. Microbiol. 50:1238-1243 (1985)).

The microbial hosts selected for the production of 2-butanol should betolerant to 2-butanol and should be able to convert carbohydrates to2-butanol using the introduced biosynthetic pathway. The criteria forselection of suitable microbial hosts include the following: intrinsictolerance to 2-butanol, high rate of carbohydrate utilization,availability of genetic tools for gene manipulation, and the ability togenerate stable chromosomal alterations.

Suitable host strains with a tolerance for 2-butanol may be identifiedby screening based on the intrinsic tolerance of the strain. Theintrinsic tolerance of microbes to 2-butanol may be measured bydetermining the concentration of 2-butanol that is responsible for 50%inhibition of the growth rate (IC50) when grown in a minimal medium. TheIC50 values may be determined using methods known in the art. Forexample, the microbes of interest may be grown in the presence ofvarious amounts of 2-butanol and the growth rate monitored by measuringthe optical density at 600 nanometers. The doubling time may becalculated from the logarithmic part of the growth curve and used as ameasure of the growth rate. The concentration of 2-butanol that produces50% inhibition of growth may be determined from a graph of the percentinhibition of growth versus the 2-butanol concentration. Preferably, thehost strain should have an IC50 for 2-butanol of greater than about0.5%. More suitable is a host strain with an IC50 for 2-butanol that isgreater than about 1.5%. Particularly suitable is a host strain with anIC50 for 2-butanol that is greater than about 2.5%.

The microbial host for 2-butanol production should also utilize glucoseand/or other carbohydrates at a high rate. Most microbes are capable ofutilizing carbohydrates. However, certain environmental microbes cannotefficiently use carbohydrates, and therefore would not be suitablehosts.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. Modes of gene transfertechnology that may be used include by electroporation, conjugation,transduction or natural transformation. A broad range of hostconjugative plasmids and drug resistance markers are available. Thecloning vectors used with an organism are tailored to the host organismbased on the nature of antibiotic resistance markers that can functionin that host.

The microbial host also may be manipulated in order to inactivatecompeting pathways for carbon flow by inactivating various genes. Thisrequires the availability of either transposons or chromosomalintegration vectors to direct inactivation. Additionally, productionhosts that are amenable to chemical mutagenesis may undergo improvementsin intrinsic 2-butanol tolerance through chemical mutagenesis and mutantscreening.

Based on the criteria described above, suitable microbial hosts for theproduction of 2-butanol include, but are not limited to, members of thegenera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferredhosts include: Escherichia coli, Alcaligenes eutrophus, Bacilluslicheniformis, Paenibacillus macerans, Rhodococcus erythropolis,Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium,Enterococcus gallinarium, Enterococcus faecalis, Pediococcuspentosaceus, Pediococcus acidilactici, Bacillus subtilis andSaccharomyces cerevisiae.

Construction of Production Host

Recombinant organisms containing the necessary genes that encode theenzymatic pathway for the conversion of a fermentable carbon substrateto 2-butanol may be constructed using techniques well known in the art.Genes encoding the enzymes of, for example, the 2-butanol biosyntheticPathway 1: acetolactate synthase, acetolactate decarboxylase, acetoinaminase (or amine:pyruvate transaminase), aminobutanol kinase,aminobutanol O-phosphate lyase and butanol dehydrogenase may be isolatedfrom various sources, as described above.

Methods of obtaining desired genes from a bacterial genome are commonand well known in the art of molecular biology. For example, if thesequence of the gene is known, primers may be designed and the desiredsequence amplified using standard primer-directed amplification methodssuch as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtainamounts of DNA suitable for cloning into expression vectors. If a genethat is heterologous to a known sequence is to be isolated, suitablegenomic libraries may be created by restriction endonuclease digestionand may be screened with probes having complementary sequence to thedesired gene sequence. Once the sequence is isolated, the DNA may beamplified using standard primer-directed amplification methods such aspolymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts ofDNA suitable for cloning into expression vectors, which are thentransformed into appropriate host cells.

In addition, given the amino acid sequence of a protein with desiredenzymatic activity, the coding sequence may be ascertained by reversetranslating the protein sequence. A DNA fragment containing the codingsequence may be prepared synthetically and cloned into an expressionvector, then transformed into the desired host cell.

In preparing a synthetic DNA fragment containing a coding sequence, thissequence may be optimized for expression in the target host cell. Toolsfor codon optimization for expression in a heterologous host are readilyavailable. Some tools for codon optimization are available based on theGC content of the host organism. The GC contents of some exemplarymicrobial hosts are given Table 3.

TABLE 3 GC Contents of Microbial Hosts Strain % GC B. licheniformis 46B. subtilis 42 C. acetobutylicum 37 E. coli 50 P. putida 61 A. eutrophus61 Paenibacillus macerans 51 Rhodococcus erythropolis 62 Brevibacillus50 Paenibacillus polymyxa 50

Once the relevant pathway genes are identified and isolated they may betransformed into suitable expression hosts by means well known in theart. Vectors useful for the transformation of a variety of host cellsare common and commercially available from companies such as EPICENTRE®(Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.), Stratagene (LaJolla, Calif.), and New England Biolabs, Inc. (Beverly, Mass.).Typically the vector contains a selectable marker and sequences allowingautonomous replication or chromosomal integration in the desired host.In addition, suitable vectors comprise a promoter region which harborstranscriptional initiation controls and a transcriptional terminationcontrol region, between which a coding region DNA fragment may beinserted, to provide expression of the inserted coding region. Bothcontrol regions may be derived from genes homologous to the transformedhost cell, although it is to be understood that such control regions mayalso be derived from genes that are not native to the specific specieschosen as a production host.

Initiation control regions or promoters, which are useful to driveexpression of the relevant pathway coding regions in the desired hostcell are numerous and familiar to those skilled in the art. Virtuallyany promoter capable of driving these genetic elements is suitable foruse including, but not limited to, promoters derived from the followinggenes: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1,URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, and GPM (useful for expression inSaccharomyces); AOX1 (useful for expression in Pichia); as well as thelac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc promoters (usefulfor expression in Escherichia coli, Alcaligenes, and Pseudomonas); theamy, apr, and npr promoters, and various phage promoters useful forexpression in Bacillus subtilis, Bacillus licheniformis, andPaenibacillus macerans; nisA (useful for expression Gram-positivebacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769(1998)); and the synthetic P11 promoter (useful for expression inLactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)).

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

Certain vectors are capable of replicating in a broad range of hostbacteria and can be transferred by conjugation. The complete andannotated sequence of pRK404 and three related vectors: pRK437, pRK442,and pRK442 (H), are available. These derivatives have proven to bevaluable tools for genetic manipulation in Gram-negative bacteria (Scottet al., Plasmid 50(1):74-79 (2003)). Several plasmid derivatives ofbroad-host-range Inc P4 plasmid RSF1010 are also available withpromoters that can function in a range of Gram-negative bacteria.Plasmid pAYC36 and pAYC37, have active promoters along with multiplecloning sites to allow for heterologous gene expression in Gram-negativebacteria.

Chromosomal gene replacement tools are also widely available. Forexample, a thermosensitive variant of the broad-host-range repliconpWV101 has been modified to construct a plasmid pVE6002 which can beused to effect gene replacement in a range of Gram-positive bacteria(Maguin et al., J. Bacteriol. 174(17):5633-5638 (1992)). The expressionof a 2-butanol biosynthetic pathway in various preferred microbial hostsis described in more detail below.

Expression of a 2-butanol Biosynthetic Pathway in E. coli

Vectors useful for the transformation of E. coli are common andcommercially available from the companies listed above. For example, thegenes of a 2-butanol biosynthetic pathway may be isolated from varioussources, as described above, cloned onto a modified pUC19 vector andtransformed into E. coli NM522, as described in Examples 10 and 11.Alternatively, the genes encoding a 2-butanol biosynthetic pathway maybe divided into multiple operons, cloned onto expression vectors, andtransformed into various E. coli strains, as described in Examples 13,15, and 15.

Expression of a 2-butanol Biosynthetic Pathway in Rhodococcuserythropolis

A series of E. coli-Rhodococcus shuttle vectors are available forexpression in R. erythropolis, including, but not limited to pRhBR17 andpDA71 (Kostichka et al., Appl. Microbiol. Biotechnol. 62:61-68 (2003)).Additionally, a series of promoters are available for heterologous geneexpression in R. erythropolis (see for example Nakashima et al., Appl.Environ. Microbiol. 70:5557-5568 (2004), and Tao et al., Appl.Microbiol. Biotechnol. 2005, DOI 10.1007/s00253-005-0064). Targeted genedisruptions in chromosomal genes of R. erythropolis may be created usingthe methods described by Tao et al., supra, and Brans et al. (Appl.Envion. Microbiol. 66: 2029-2036 (2000)).

The heterologous genes required for the production of 2-butanol, asdescribed above, may be cloned initially in pDA71 or pRhBR71 andtransformed into E. coli. The vectors may then be transformed into R.erythropolis by electroporation, as described by Kostichka et al.,supra. The recombinants may be grown in synthetic medium containingglucose and the production of 2-butanol can be followed usingfermentation methods known in the art.

Expression of a 2-butanol Biosynthetic Pathway in B. Subtilis

Methods for gene expression and creation of mutations in B. subtilis arealso well known in the art. For example, the genes of a 2-butanolbiosynthetic pathway may be isolated from various sources, as describedabove, cloned into a modified E. coli-Bacillus shuttle vector andtransformed into Bacillus subtilis BE1010, as described in Example 12,The desired genes may be cloned into a Bacillus expression vector andtransformed into a strain to make a production host. Alternatively, thegenes may be integrated into the Bacillus chromosome using conditionalreplicons or suicide vectors that are known to one skilled in the art.For example, the Bacillus Genetic Stock Center carries numerousintegration vectors.

Expression of a 2-butanol Biosynthetic Pathway in B. licheniformis

Most of the plasmids and shuttle vectors that replicate in B. subtilismay be used to transform B. licheniformis by either protoplasttransformation or electroporation. The genes required for the productionof 2-butanol may be cloned in plasmids pBE20 or pBE60 derivatives(Nagarajan et al., Gene 114:121-126 (1992)). Methods to transform B.licheniformis are known in the art (for example see Fleming et al. Appl.Environ. Microbiol., 61(11):3775-3780 (1995)). The plasmids constructedfor expression in B. subtilis may be transformed into B. licheniformisto produce a recombinant microbial host that produces 2-butanol.

Expression of a 2-butanol Biosynthetic Pathway in Paenibacillus macerans

Plasmids may be constructed as described above for expression in B.subtilis and used to transform Paenibacillus macerans by protoplasttransformation to produce a recombinant microbial host that produces2-butanol.

Expression of a 2-butanol Biosynthetic Pathway in Alcaligenes(Ralstonia) eutrophus

Methods for gene expression and creation of mutations in Alcaligeneseutrophus are known in the art (see for example Taghavi et al., Appl.Environ. Microbiol., 60(10):3585-3591 (1994)). The genes for a 2-butanolbiosynthetic pathway may be cloned in any of the broad host rangevectors described above, and electroporated into Alcaligenes eutrophusto generate recombinants that produce 2-butanol. Thepoly(hydroxybutyrate) pathway in Alcaligenes has been described indetail, a variety of genetic techniques to modify the Alcaligeneseutrophus genome are known, and those tools can be applied forengineering a 2-butanol biosynthetic pathway.

Expression of a 2-butanol Biosynthetic Pathway in Pseudomonas putida

Methods for gene expression in Pseudomonas putida are known in the art(see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which isincorporated herein by reference). The genes of a 2-butanol biosyntheticpathway may be inserted into pPCU18, and this ligated DNA may beelectroporated into electrocompetent Pseudomonas putida DOT-T1 C5aAR1cells to generate recombinants that produce 2-butanol.

Expression of a 2-butanol Biosynthetic Pathway in Lactobacillusplantarum

The Lactobacillus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Bacillus subtilis andStreptococcus may be used for Lactobacillus. Non-limiting examples ofsuitable vectors include pAMβ1 and derivatives thereof (Renault et al.,Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231(1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl.Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative plasmid(Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520(Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997));pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001));and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903(1994)). Several plasmids from Lactobacillus plantarum have also beenreported (van Kranenburg et al., Appl. Environ. Microbiol.71(3):1223-1230 (2005)).

The various genes for a 2-butanol biosynthetic pathway may be assembledinto any suitable vector, such as those described above. The codons canbe optimized for expression based on the codon index deduced from thegenome sequences of Lactobacillus plantarum or Lactobacillusarizonensis. The plasmids may be introduced into the host cell usingmethods known in the art, such as electroporation (Cruz-Rodz et al.Molecular Genetics and Genomics 224:1252-154 (1990), Bringel, et al.Appl. Microbiol. Biotechnol. 33: 664-670 (1990), Alegre et al., FEMSMicrobiology letters 241:73-77 (2004)), and conjugation (Shrago et al.,Appl. Environ. Microbiol. 52:574-576 (1986)). The 2-butanol biosyntheticpathway genes can also be integrated into the chromosome ofLactobacillus using integration vectors (Hols et al., Appl. Environ.Microbiol. 60:1401-1403 (1990), Jang et al., Micro. Lett. 24:191-195(2003)).

Expression of a 2-butanol Biosynthetic Pathway in Enterococcus faecium,Enterococcus gallinarium, and Enterococcus faecalis

The Enterococcus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Lactobacillus,Bacillus subtilis, and Streptococcus, described above, may be used forEnterococcus. Expression vectors for E. faecalis using the nisA genefrom Lactococcus may also be used (Eichenbaum et al., Appl. Environ.Microbiol. 64:2763-2769 (1998). Additionally, vectors for genereplacement in the E. faecium chromosome may be used (Nallaapareddy etal., Appl. Environ. Microbiol. 72:334-345 (2006)).

The various genes for a 2-butanol biosynthetic pathway may be assembledinto any suitable vector, such as those described above. The codons canbe optimized for expression based on the codon index deduced from thegenome sequences of Enterococcus faecalis or Enterococcus faecium. Theplasmids may be introduced into the host cell using methods known in theart, such as electroporation, as described by Cruz-Rodz et al.(Molecular Genetics and Genomics 224:1252-154 (1990)) or conjugation, asdescribed by Tanimoto et al. (J. Bacteriol. 184:5800-5804 (2002)) andGrohamann et al. (Microbiol. Mol. Biol. Rev. 67:277-301 (2003)).

Expression of a 2-butanol Biosynthetic Pathway in Pediococcuspentosaceus and Pediococcus acidilactici

The Pediococcus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Bacillus subtilis andStreptococcus, described above, may be used for Pediococcus. Anon-limiting example of a suitable vector is pHPS9 (Bukhtiyarova et al.Appl. Environ. Microbiol. 60:3405-3408 (1994)). Several plasmids fromPediococcus have also been reported (Alegre et al., FEMS Microbiol.Lett. 250:151-156 (2005); Shareck et al. Crit. Rev Biotechnol.24:155-208 (2004)).

The genes for a 2-butanol biosynthetic pathway may be assembled into anysuitable vector, such as those described above. The codons can beoptimized for expression based on the codon index deduced from thegenome sequence of Pediococcus pentosaceus. The plasmids may beintroduced into the host cell using methods known in the art, such aselectroporation (see for example, Osmanagaoglu et al., J. BasicMicrobiol. 40:233-241 (2000); Alegre et al., FEMS Microbiol. Lett.250:151-156 (2005)) and conjugation (Gonzalez and Kunka, Appl. Environ.Microbiol. 46:81-89 (1983)). The 2-butanol biosynthetic pathway genescan also be integrated into the chromosome of Pediococcus usingintegration vectors (Davidson et al. Antonie van Leeuwenhoek 70:161-183(1996)).

Fermentation Media

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include but are not limited tomonosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated. Inaddition to one and two carbon substrates, methylotrophic organisms arealso known to utilize a number of other carbon containing compounds suchas methylamine, glucosamine and a variety of amino acids for metabolicactivity. For example, methylotrophic yeasts are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion et al.,Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention,preferred carbon substrates are glucose, fructose, and sucrose. Sucrosemay be derived from renewable sugar sources such as sugar cane, sugarbeets, cassava, sweet sorghum, and mixtures thereof. Glucose anddextrose may be derived from renewable grain sources throughsaccharification of starch based feedstocks including grains such ascorn, wheat, rye, barley, oats, and mixtures thereof. In addition,fermentable sugars may be derived from renewable cellulosic orlignocellulosic biomass through processes of pretreatment andsaccharification, as described, for example, in co-owned and co-pendingU.S. Patent Application Publication No. 2007/0031918A1, which is hereinincorporated by reference. Biomass refers to any cellulosic orlignocellulosic material and includes materials comprising cellulose,and optionally further comprising hemicellulose, lignin, starch,oligosaccharides and/or monosaccharides. Biomass may also compriseadditional components, such as protein and/or lipid. Biomass may bederived from a single source, or biomass can comprise a mixture derivedfrom more than one source; for example, biomass may comprise a mixtureof corn cobs and corn stover, or a mixture of grass and leaves. Biomassincludes, but is not limited to, bioenergy crops, agricultural residues,municipal solid waste, industrial solid waste, sludge from papermanufacture, yard waste, wood and forestry waste. Examples of biomassinclude, but are not limited to, corn grain, corn cobs, crop residuessuch as corn husks, corn stover, grasses, wheat, wheat straw, barley,barley straw, hay, rice straw, switchgrass, waste paper, sugar canebagasse, sorghum, soy, components obtained from milling of grains,trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, flowers, animal manure, and mixtures thereof.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of an enzymatic pathway necessary for2-butanol production.

Culture Conditions with Temperature Lowering

In the present method, the recombinant microbial production host whichproduces 2-butanol is seeded into a fermentation medium comprising afermentable carbon substrate to create a fermentation culture. Theproduction host is grown in the fermentation culture at a firsttemperature for a first period of time. The first temperature istypically from about 25° C. to about 40° C.

Suitable fermentation media in the present invention include commoncommercially prepared media such as Luria Bertani (LB) broth, SabouraudDextrose (SD) broth or Yeast Medium (YM) broth. Other defined orsynthetic growth media may also be used, and the appropriate medium forgrowth of the particular microorganism will be known by one skilled inthe art of microbiology or fermentation science. The use of agents knownto modulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′:3′-monophosphate, may also be incorporated into thefermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0,where pH 6.0 to pH 8.0 is preferred as the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions,where anaerobic or microaerobic conditions are preferred.

The first period of time to grow the production host at the firsttemperature may be determined in a variety of ways. For example, duringthis period of growth a metabolic parameter of the fermentation culturemay be monitored. The metabolic parameter that is monitored may be anyparameter known in the art, including, but not limited to the opticaldensity, pH, respiratory quotient, fermentable carbon substrateutilization, CO₂ production, and 2-butanol production. During thisperiod of growth, additional fermentable carbon substrate may be added,the pH may be adjusted, oxygen may be added for aerobic cells, or otherculture parameters may be adjusted to support the metabolic activity ofthe culture. Though nutrients and culture conditions are supportive ofgrowth, after a period of time the metabolic activity of thefermentation culture decreases as determined by the monitored parameterdescribed above. For example, a decrease in metabolic activity may beindicated by a decrease in one or more of the following parameters: rateof optical density change, rate of pH change, rate of change inrespiratory quotient (if the host cells are aerobic), rate offermentable carbon substrate utilization, rate of 2-butanol production,rate of change in CO₂ production, or rate of another metabolicparameter. The decrease in metabolic activity is related to thesensitivity of the host cells to the production of 2-butanol and/or thepresence of 2-butanol in the culture. When decreased metabolic activityis detected, the temperature of the fermentation culture is lowered toreduce the sensitivity of the host cells to 2-butanol and thereby allowfurther production of 2-butanol. In one embodiment, the lowering of thetemperature coincides with a change in the metabolic parameter that ismonitored.

In one embodiment, the change in metabolic activity is a decrease in therate of 2-butanol production. 2-Butanol production may be monitored byanalyzing the amount of 2-butanol present in the fermentation culturemedium as a function of time using methods well known in the art, suchas using high performance liquid chromatography (HPLC) or gaschromatography (GC), which are described in the Examples herein. GC ispreferred due to the short assay time.

Alternatively, the lowering of the temperature of the fermentationculture may occur at a predetermined time. The first period of time maybe predetermined by establishing a correlation between a metabolicparameter of the fermentation culture and time in a series of testfermentations runs. A correlation between a metabolic parameter, asdescribed above, and time of culture growth may be established for any2-butanol producing host by one skilled in the art. The specificcorrelation may vary depending on conditions used including, but notlimited to, carbon substrate, fermentation conditions, and the specificrecombinant 2-butanol producing microbial production host. Thecorrelation is most suitably made between 2-butanol production orspecific glucose consumption rate and time of culture growth. Once thepredetermined time has been established from the correlation, thetemperature of the fermentation culture in subsequent fermentation runsis lowered at the predetermined time. For example, if it is determinedby monitoring a metabolic parameter in the test fermentation runs thatthe rate of production of 2-butanol decreases after 12 hours, thetemperature in subsequent fermentations runs is lowered after 12 hourswithout the need to monitor 2-butanol production in the subsequent runs.

After the first period of time, the temperature of the fermentationculture is lowered to a second temperature. Typically, the secondtemperature is about 3° C. to about 25° C. lower than the firsttemperature. Reduction in temperature to enhance tolerance of the hostcells to 2-butanol is balanced with maintaining the temperature at alevel where the cells continue to be metabolically active for 2-butanolproduction. For example, a fermentation culture that has been grown atabout 35° C. may be reduced in temperature to about 28° C.; or a culturegrown at about 30° C. may be reduced in temperature to about 25° C. Thechange in temperature may be done gradually over time or may be made asa step change. The production host is incubated at the secondtemperature for a second period of time, so that 2-butanol productioncontinues. The second period of time may be determined in the samemanner as the first period of time described above, e.g., by monitoringa metabolic parameter or by using a predetermined time.

Additionally, the temperature lowering and incubation steps may berepeated one or more times to more finely balance metabolic activity for2-butanol production and 2-butanol sensitivity. For example, a culturethat has been grown at about 35° C. may be reduced in temperature toabout 32° C., followed by an incubation period. During this period ametabolic parameter of the fermentation culture may be monitored asdescribed above, or a predetermined time may be used. It is particularlysuitable to monitor the production of 2-butanol during this incubationperiod. When monitoring indicates a decrease in metabolic activity or ata predetermined time, the temperature may be reduced a second time. Forexample, the temperature may be reduced from about 32° C. to about 28°C. The temperature lowering and incubation steps may be repeated a thirdtime where the temperature is reduced, for example, to about 20° C. Theproduction host is incubated at the lowered temperature so that2-butanol production continues. The steps may be repeated further asnecessary to obtain the desired 2-butanol titer.

Industrial Batch and Continuous Fermentations

The present process employs a batch method of fermentation. A classicalbatch fermentation is a closed system where the composition of themedium is set at the beginning of the fermentation and not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the medium is inoculated with the desired organismor organisms, and fermentation is permitted to occur without addinganything to the system. Typically, however, a “batch” fermentation isbatch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the fed-batch system.Fed-batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in fed-batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and fed-batch fermentations are common and well knownin the art and examples may be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, MukundV., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated byreference.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation medium is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by the turbidity of the culture medium, is kept constant.Continuous systems strive to maintain steady state growth conditions andthus the cell loss due to the medium being drawn off must be balancedagainst the cell growth rate in the fermentation. Methods of modulatingnutrients and growth factors for continuous fermentation processes aswell as techniques for maximizing the rate of product formation are wellknown in the art of industrial microbiology and a variety of methods aredetailed by Brock, supra.

It is contemplated that the present invention may be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for 2-butanol production.

Methods for 2-Butanol Isolation from the Fermentation Medium

The bioproduced 2-butanol may be isolated from the fermentation mediumusing methods known in the art for ABE fermentations (see for Example,Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al.,Process Biochem. 27:61-75 (1992), and references therein). For example,solids may be removed from the fermentation medium by centrifugation,filtration, decantation, or the like. Then, the 2-butanol may beisolated from the fermentation medium using methods such asdistillation, azeotropic distillation, liquid-liquid extraction,adsorption, gas stripping, membrane evaporation, or pervaporation.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating a preferredembodiment of the invention, are given by way of illustration only. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques described inthe Examples are well known in the art and are described by Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.,(1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials described for the growth and maintenance of bacterial cellswere obtained from Aldrich Chemicals (Milwaukee, Wis.), BD DiagnosticSystems (Sparks, Md.), Life Technologies (Rockville, Md.), or SigmaChemical Company (St. Louis, Mo.) unless otherwise specified. Bacterialstrains are obtained from the American Type Culture Collection (ATCC,Manassas, Va.) unless otherwise noted.

Oligonucleotide Primers Described in the Following Examples are Given in

TABLE 4 All oligonucleotide primers were synthesized by Sigma-Genosys(Woodlands, TX).Table 4 Cloning and Screening Primers SEQ Primer ID GeneName Sequence NO: Description budB B1 CACCATGGACAAACAGTA 15 budBTCCGGTACGCC forward budB B2 CGAAGGGCGATAGCTTTA 16 budB CCAATCC reversebudA B3 CACCATGAATCATTCTGC 17 budA TGAATGCACCTGCG forward budA B4GATACTGTTTGTCCATGT 18 budA GACC reverse budC B5 CACCATGAAAAAAGTCGC 19budC ACTTGTTACC forward budC B6 TTAGTTAAATACCAT 20 budC reverse pddA B7CACCATGAGATCGA 21 pddABC AAAGATTTG forward pddC B8 CTTAGAGAAGTTAATCGT 22pddABC CGCC reverse sadh B9 CACCATGAAAGCCCTCCA 23 sadh GTACACC forwardsadh B10 CGTCGTGTCATGCCCGG 24 sadh G reverse budA B11GATCGAATTCGTTTAAACT 25 budABC TAGTTTTCTACCGCACG forward budC B12GATCGCATGCAAGCTTTC 26 budABC ATATAGTCGGAATTCC reverse pddA B13GATCGAATTCGTTTAAACA 27 pddABC AAGGAGGTCTGATTCATG forward AGATCG pddC B14GATCGGATTCTTAATCGT 28 pddABC CGCC reverse sadh B15 GATCGGATCCAAAGGAGG 29sadh TCGGGCGCATGAAAGCC forward C sadh B16 GATCTCTAGAAAGCTTTC 30 sadhAGCCCGGGACGACC reverse — BenF ACTTTCTTTCGCCTGTTTC 31 — AC — BenBPRCATGAAGCTTGTTTAAACT 32 — CGGTGACCTTGAAAATAA TGAAAACTTATATTGTTTTGAAAATAATGAAAACTTAT ATTG budAB BABC F GAGCTCGAATTCAAAGGA 33 budABGGAAGTGTATATGAATCA forward TTC budAB BAB R GGATCCTCTAGAATTAGT 34 budABTAAATACCATCCCGCCG reverse budC BC Spe ACTAGTAAAGGAGGAAAG 40 budC FAGTATGAAGAAGGTCGCA forward CT budC BC Xba TCTAGAAAGCAGGGGCAA 41 budC RGCCATGTC reverse pddAB DDo AAGCTTAAAGGAGGCTGA 44 pddABC- C- ForTTCATGAGATCGAAAAGA ddrAB ddrAB TT forward pddAB DDo TCTAGATTATTCATCCTGC45 pddABC- C- Rev TGTTCTCC ddrAB ddrAB reverse chnA ChnA FCATCAATTGACTACGTAG 54 chnA TCGTACGTGTAAGGAGGT forwardTTGAAATGGAAAAAATTAT G chnA ChnA R CATGCTAGCCCCGGGTAT 55 chnACTTCTACTCATTTTTTATTT reverse CG — Top CTAGAAGTCAAAAGCCTC 58 forward terF1 CGACCGGAGGCTTTTGA — Top CTGCTCGAGTTGCTAGC 59 forward ter F2AAGTTTAAACAAAAAAAA GCCCGCTCATTAGGCGG GCTGAGCT — Bot CAGCCCGCCTAATGAGC 60reverse ter R1 GGGCTTTTTTTTGTTTAA AC — Bot TTGCTAGCAACTCGAGCA 61 reverseter R2 GTCAAAAGCCTCCGGTC GGAGGCTTTTGACTT KA-AT OT872 CTCCGGAATTCATGTCTG127 Aminoalco- ACGGACGACTCACCGCA hol kinase/ lyase oper- on forwardKA-AT OT873 TTCCAATGCATTGGCTGC 128 Aminoalco- AGTTATCTCTGTGCACGA holkinase/ GTGCCGATGA lyase oper- on reverse KA OT879 AACAGCCAAGCTTGGCT 129Aminoalco- GCAGTCATCGCGCATTCT hol kinase CCGGG reverse AT OT880TCTCCGGAATTCATGACG 130 Aminoalco- TCTGAAATGACAGCGACA hol lyase GAAGforward pBAD. OT909 GCTAACAGGAGGAAGAA 131 Adds EcoRI HisBTTCATGGGGGGTTCTC site to replace NcoI site pBAD. OT910GAGAACCCCCCATGAATT 132 Adds EcoRI HisB CTTCCTCCTGTTAGC site to replaceNcoI site BudAB N84seqR3 GGACCTGCTTCGCTTTAT 159 reverse CG APT APTforGCGCGCCCGGGAAGAAG 162 APT forward GAGCTCTTCACCATGAAC AAACCACAGTCTTGG APTAPTrev GCGCGCCCGGGTTCATG 163 APT reverse CCACCTCTGCG

TABLE 5 Sequencing Primers SEQ Gene- ID Name Sequence specific NO: M13Forward GTAAAACGACGGCCAGT — 35 M13 Reverse AACAGCTATGACCATG — 36 N83SeqF2 GCTGGATTACCAGCTCGACC — 37 N83 SeqF3 CGGACGCATTACCGGCAAAG — 38 N84SeqR2 GCATCGAGATTATCGGGATG — 65 N84 SeqR4 CGAAGCGAGAGAAGTTATCC — 39 TrcF TTGACAATTAATCATCCGGC all 42 Trc R CTTCTCTCATCCGCCAAAAC all 43 DDko seqF2 GCATGGCGCGGATTTGACGAAC pddABC- 46 ddrAB DDko seq F5CATTAAAGAGACCAAGTACGTG pddABC- 47 ddrAB DDko seq F7ATATCCTGGTGGTGTCGTCGGCGT pddABC- 48 ddrAB DDko seq F9TCTTTGTCACCAACGCCCTGCG pddABC- 49 ddrAB DDko seq R1GCCCACCGCGCTCGCCGCCGCG pddABC- 50 ddrAB DDko seq R3CCCCCAGGATGGCGGCTTCGGC pddABC- 51 ddrAB DDko seq R7GGGCCGACGGCGATAATCACTT pddABC- 52 ddrAB DDko seq R10TTCTTCGATCCACTCCTTAACG pddABC- 53 ddrAB chnSeq F1 CTCAACAGGGTGTAAGTGTAGTchnA 56 chnSeq R1 CGTTTTGATATAGCCAGGATGT chnA 57 pCL1925 vec FCGGTATCATCAACAGGCTTACC all 62 pCL1925 vec R1 AGGGTTTTCCCAGTCACGACGT all63 pCL1925 vec R2 CGCAATAGTTGGCGAAGTAATC all 64 APTseqRevGCTAGAGATGATAGC APT 160 APTseqFor GGAAGAGACTATCCAGCG APT 161Methods for Determining 2-Butanol and 2-Butanone Concentration inCulture Media

The concentration of 2-butanol and 2-butanone in the culture media canbe determined by a number of methods known in the art. For example, aspecific high performance liquid chromatography (HPLC) method utilized aShodex SH-1011 column with a Shodex SH-G guard column, both purchasedfrom Waters Corporation (Milford, Mass.), with refractive index (RI)detection. Chromatographic separation was achieved using 0.01 M H₂SO₄ asthe mobile phase with a flow rate of 0.5 mL/min and a column temperatureof 50° C. Under the conditions used, 2-butanone and 2-butanol hadretention times of 39.5 and 44.3 min, respectively. Alternatively, gaschromatography (GC) methods are available. For example, a specific GCmethod utilized an HP-INNOWax column (30 m×0.53 mm id, 1 μm filmthickness, Agilent Technologies, Wilmington, Del.), with a flameionization detector (FID). The carrier gas was helium at a flow rate of4.5 mL/min, measured at 150° C. with constant head pressure; injectorsplit was 1:25 at 200° C.; oven temperature was 45° C. for 1 min, 45 to220° C. at 10° C./min, and 220° C. for 5 min; and FID detection wasemployed at 240° C. with 26 mL/min helium makeup gas. The retentiontimes of 2-butanone and 2-butanol were 3.61 and 5.03 min, respectively.

2-Butanone can also be detected by derivatization with3-methyl-2-benzothiazolinone hydrazone (MBTH). An aqueous solutioncontaining 2-butanone is mixed with an equal volume of an aqueoussolution of 6 mg/mL MBTH in 375 mM glycine-HCl (pH 2.7) and incubated at100° C. for 3 min. The resulting MBTH-derivatized samples are analyzedon a 25 cm×4.6 mm (id) Supelosil LC-18-D5 5 μm column (Supelco) using amobile phase of 55% acetonitrile in water at a flow rate of 1 mL/min.The 2-butanone derivative appears as two peaks (cis and trans isomers)with retention times of approximately 12.3 and 13.3 min and absorbancemaxima of 230 and 307 nm.

The meaning of abbreviations is as follows: “s” means second(s), “min”means minute(s), “h” means hour(s), “psi” means pounds per square inch,“nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm”means nanometers, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmol” means micromole(s)”, “g” means gram(s), “μg” meansmicrogram(s) and “ng” means nanogram(s), “PCR” means polymerase chainreaction, “OD” means optical density, “OD₆₀₀” means the optical densitymeasured at a wavelength of 600 nm, “kDa” means kilodaltons, “g” meansthe gravitation constant, “bp” means base pair(s), “kbp” means kilobasepair(s), “% w/v” means weight/volume percent, % v/v” means volume/volumepercent, “wt %” means percent by weight, “nt” means not tested, “HPLC”means high performance liquid chromatography, and “GC” means gaschromatography. The term “molar selectivity” is the number of moles ofproduct produced per mole of sugar substrate consumed and is reported asa percent.

Example 1 Increased Tolerance of Lactobacillus plantarum PN0512 to1-butanol, iso-butanol and 2-butanol at Decreased Growth Temperatures

Tolerance levels of bacterial strain Lactobacillus plantarum PN0512(ATCC # PTA-7727) were determined at 25° C., 30° C. and 37° C. asfollows. The strain was cultured in S30L medium (i.e., 10 mM ammoniumsulfate, 5 mM potassium phosphate buffer, pH 7.0, 50 mM MOPS, pH 7.0, 2mM MgCl₂, 0.7 mM CaCl₂, 50 μM MnCl₂, 1 μM FeCl₃, 1 μM ZnCl₂, 1.72 μMCuCl₂, 2.53 μM CoCl₂, 2.42 μM Na₂MoO₄, 2 μM thiamine hydrochloride, 10mM glucose, and 0.2% yeast extract). An overnight culture in the absenceof any test compound was started in 15 mL of the S30L medium in a 150 mLflask, with incubation at 37° C. in a shaking water bath. The nextmorning, the overnight culture was diluted into three 500 mL flaskscontaining 150 mL of fresh medium to an initial OD₆₀₀ of about 0.08.Each flask was incubated in a shaking water bath, one each at 25° C.,30° C. and 37° C. Each large culture was allowed to acclimate at thetest temperature for at least 0.5 h. After the acclimation period, eachlarge culture was split into flasks in the absence (control) and in thepresence of various amounts of 1-butanol, isobutanol or 2-butanol, aslisted in Tables 6, 7, and 8, respectively. Growth was followed bymeasuring OD₆₀₀ for six hours after addition of the compounds. Theresults are summarized in Tables 6, 7, and 8 below.

TABLE 6 Growth of L. plantarum PN0512 in the presence of 1-butanol atdifferent temperatures Concentration 1- butanol (% w/v) 37° C. 30° C.25° C. 0.0 +¹ + + 1.0 + nt³ nt 1.2 + nt nt 1.4 + nt nt 1.5 + + + 1.6 +nt nt 1.8 + nt nt 2.0 + + + 2.1 + nt nt 2.2 + nt nt 2.3 + nt nt 2.4−² + + 2.5 − nt nt 2.7 − + nt 2.9 − − + 3.1 − − + 3.2 nt − − 3.3 nt nt −3.4 nt − − ¹“+” = growth observed as an increase in OD₆₀₀. ²“−” = nogrowth observed, i.e. no change in OD₆₀₀. ³“nt” = not tested

TABLE 7 Growth of L. plantarum PN0512 in the presence of isobutanol atdifferent temperatures Concentration isobutanol (% w/v) 37° C. 30° C.25° C. 0.0 +¹ + + 0.5 + nt³ nt 1.0 + nt nt 1.5 + + + 1.6 + nt nt 1.8 +nt nt 2.0 + + + 2.1 + nt nt 2.3 + nt nt 2.4 + + + 2.5 + nt nt 2.7 + + +2.9 + + + 3.1 + + + 3.3 nt −² + 3.4 − nt nt 3.5 nt nt + 3.6 nt nt − 3.8− nt nt 4.3 − nt nt ¹“+” = growth observed as an increase in OD₆₀₀. ²“−”= no growth observed, i.e. no change in OD₆₀₀. ³“nt” = not tested

TABLE 8 Growth of L. plantarum PN0512 in the presence of 2-butanol atdifferent temperatures Concentration 2- butanol (% w/v) 37° C. 30° C.25° C. 0.0 +¹ + + 1.8 + nt³ nt 2.1 + nt nt 2.5 + nt nt 2.9 + + + 3.1 +nt nt 3.5 + nt nt 3.6 + nt nt 3.8 + + + 4.0 nt + nt 4.3 + + + 4.5 −² +nt 4.7 − + + 4.9 nt − + 5.2 − nt + 5.6 − nt − 6.0 − nt nt 6.4 − nt nt7.3 − nt nt ¹“+” = growth observed as an increase in OD₆₀₀. ²“−” = nogrowth observed, i.e. no change in OD₆₀₀. ³“nt” = not tested

All three butanols showed a similar effect of temperature on growthinhibition of L. plantarum PN0512. The concentration that resulted infull growth inhibition was greater at 25° C. than at 37° C. In the caseof 1-butanol, growth was observed at 37° C. in 2.3% 1-butanol, but not2.4%. However, at 30° C. growth was observed in 2.7%, but not 2.9%, andat 25° C. growth was observed even in 3.1% 1-butanol. Thus, theconcentration of 1-butanol that completely inhibited growth increased asgrowth temperature decreased. Likewise, in the case of isobutanol,growth was observed in 3.5% at 25° C. while growth was observed in 3.1%at 30° C. and 37° C., but not in 3.3% or 3.4%. Similarly, in the case of2-butanol growth was observed at 37° C. in 4.3%, but not in 4.5%; at 30°C. in 4.7%, but not in 4.9%; and at 25° C. in 5.2%. Thus the toleranceof L. plantarum PN0512 to butanols increased with decreased growthtemperature.

Example 2 Increased Tolerance of Escherichia coli to 1-butanol atDecreased Exposure Temperature

The effect of growth and exposure temperature on survival of Escherichiacoli in the presence of 1-butanol was tested using stationary phasecultures in a rich medium and log phase cultures in a defined medium.For the stationary phase studies, E. coli strain MG1655 (ATCC #700926)was grown overnight in LB medium (Teknova, Half Moon Bay, Calif.) withshaking at 250 rpm at 42° C., 29° C. or 28° C. Survival of 1-butanolshock was tested at exposure temperatures of 0° C., 28° C. or 42° C. The1-butanol exposure at 28° C. or 42° C. was started immediately afterremoving the overnight cultures from the growth incubators. The1-butanol exposure at 0° C. was done after allowing the overnightcultures to cool on ice for about 15 min. A series of solutions of1-butanol at different concentrations in LB medium was made and 90 μLaliquots were put in microfuge tubes. To these were added 10 μL of theovernight cultures and the tubes were immediately placed in shakingincubators at 42° C. or 28° C. or left on ice for 30 min. To stop theeffect of 1-butanol on the cultures, a 10⁻² dilution was done by placing2 μL of the treated culture into 198 μL of LB medium in wells of amicroplate. Then, 5 μL of the undiluted treated cultures were spotted onLB agar plates. Subsequent 10-fold serial dilutions of 10⁻³, 10⁻⁴, 10⁻⁵and 10⁻⁶ of the exposed cultures were done by serial pipetting of 20 μL,starting with the 10⁻² dilution cultures, into 180 μL of LB medium inthe microplate, using a multi-channel pipette. Prior to each transfer,the cultures were mixed by pipetting up and down six times. Eachdilution (5 μL) was spotted onto an LB plate using a multi-channelpipette and allowed to soak into the plate. The plates were inverted andincubated overnight at 37° C. The number of colonies for each dilutionwas counted and the % growth inhibition was calculated by comparisonwith a control culture that had not been exposed to 1-butanol. Survivalof 0% was recorded when no colonies in the spots of the undiluted or anyof the serial dilutions were observed. The results are shown in Table 9.

TABLE 9 Survival of stationary phase E. coli in 1-butanol at 42° C., 28°C., or 0° C. Grown at Grown Grown Grown Grown Grown 42° C. at 29° C. at42° C. at 28° C. at 42° C. at 29° C. 1- % survival after 30 min %survival after 30 min % survival after 30 min Butanol exposure atexposure at exposure at % (w/v) 42° C. 28° C. 0° C. 1.0 100 100 100 100100 100 1.5 0.1 0.1 100 100 100 100 2.0 0 0.1 100 100 100 100 2.5 0 0100 100 100 100 3.0 0 0 100 100 100 100 3.5 0 0 3 10 100 100 4.0 0 00.0004 0.0003 100 100 5.0 nt¹ nt nt nt 1 1 6.0 nt nt nt nt 0 0.001 7.0nt nt nt nt 0 0 ¹“nt” = not tested

A similar study was done with log-phase cultures of E. coli grown in adefined medium. E. coli strain MG1655 was allowed to grow overnight inMOPS 0.2% glucose medium (Teknova, Half Moon Bay, Calif.) at 42° C. or28° C. The following day, the cultures were diluted into fresh mediumand allowed to grow at the same temperature until in the log phase ofgrowth. The OD₆₀₀ was 0.74 for the 28° C. culture and was 0.72 for the42° C. culture. Both of these log phase cultures were exposed to1-butanol at 42° C., 28° C. and 0° C. as follows. A series of solutionsof 1-butanol at different concentrations in MOPS 0.2% glucose medium wasmade and 90 μL aliquots were put in microfuge tubes. To these were added10 μL of the log phase cultures and the tubes were immediately placed inshaking incubators at 42° C. or 28° C. or left on ice for 30 min. Tostop the effect of 1-butanol on the cultures, a 10⁻² dilution was doneby placing 2 μL of the treated culture into 198 μL of LB medium in wellsof a microplate. Then 5 μL of the undiluted treated cultures werespotted on LB agar plates. Subsequent 10-fold serial dilutions of 10⁻³,10⁻⁴, 10⁻⁵ and 10⁻⁶ of the exposed cultures were done by serialpipetting of 20 μL, starting with the 10⁻² dilution cultures, into 180μL of LB medium in the microplate, using a multi-channel pipette. Priorto each transfer, the cultures were mixed by pipetting up and down sixtimes. Each dilution (5 μL) was spotted onto an LB plate using amulti-channel pipette and allowed to soak into the plate. The plateswere inverted and incubated overnight at 37° C. The number of coloniesfor each dilution was counted and the % growth inhibition was calculatedby comparison with a control culture that had not been exposed to1-butanol. Survival of 0% was recorded when no colonies in the spots ofthe undiluted or any of the serial dilutions were observed. The resultsare shown in Table 10.

TABLE 10 Survival of log-phase E. coli in 1-butanol at 42° C., 28° C.,or 0° C. Grown at Grown Grown Grown Grown Grown 42° C. at 28° C. at 42°C. at 28° C. at 42° C. at 29° C. 1- % survival after 30 min % survivalafter 30 min % survival after 30 min Butanol exposure at exposure atexposure at % (w/v) 42° C. 28° C. 0° C. 1.0 100 100 nt¹ nt nt nt 1.5 0 0100 100 nt nt 2.0 0 0 100 100 nt nt 2.5 0 0 0.1 50 100 100 3.0 0 0 0 0100 100 3.5 0 0 0.01 0 100 100 4.0 0 0 0.001 0 100 100 4.5 nt nt 0 0 100100 5.0 nt nt nt nt 10 50 6.0 nt nt nt nt 1 1 ¹“nt” = not tested

For both the stationary phase and log-phase cultures of E. coli MG1655,the growth temperature had very little, if any, effect on the survivalof a 1-butanol shock. However, the exposure temperature had a majoreffect on the survival of E. coli to 1-butanol shock. As can be seenfrom the data in Tables 9 and 10, the tolerance of E. coli MG1655 to1-butanol increased with decreasing exposure temperature.

Example 3 Increased Tolerance of Escherichia coli to 2-butanone atDecreased Exposure Temperature

The effect of exposure temperature on survival of Escherichia coli inthe presence of 2-butanone (also referred to herein as methyl ethylketone or MEK) was tested as follows. E. coli strain BW25113 (The ColiGenetic Stock Center (CGSC), Yale University; #7636) was grown overnightin LB medium (Teknova, Half Moon Bay, Calif.) with shaking at 250 rpm at37° C. Survival of MEK shock was tested at exposure temperatures of 28°C. or 37° C. A series of solutions of MEK at different concentrations inLB medium was made and 90 μL aliquots were put in microfuge tubes. Tothese were added 10 μL of the overnight culture and the tubes wereimmediately placed in shaking incubators at 37° C. or 28° C. for 30 min.To stop the effect of MEK on the cultures, a 10⁻² dilution was done byplacing 2 μL of the MEK treated culture into 198 μL of LB medium inwells of a microplate. Then 5 μL of the undiluted treated cultures werespotted on LB agar plates. Subsequent 10-fold serial dilutions of 10⁻³,10⁻⁴, 10⁻⁵ and 10⁻⁶ of the exposed cultures were done by serialpipetting of 20 μL, starting with the 10⁻² dilution cultures, into 180μL of LB medium in the microplate, using a multi-channel pipette. Priorto each transfer, the cultures were mixed by pipetting up and down sixtimes. Each dilution (5 μL) was spotted onto LB plates using amulti-channel pipette and allowed to soak into the plate. The plateswere inverted and incubated overnight at 37° C. The number of coloniesfor each dilution was counted and the % growth inhibition was calculatedby comparison with a control culture that had not been exposed to MEK.Survival of 0% was recorded when no colonies in the spots of theundiluted or any of the serial dilutions were observed. The results,given as the average of duplicate experiments, are shown in Table 11.

TABLE 11 Survival of E. coli in MEK at 37° C. and 28° C. MEK % w/v %Survival at 37° C. % Survival at 28° C. 0 100 100 4 100 100 6 0 100 8 00.002

Reducing the exposure temperature from 37° C. to 28° C. dramaticallyimproved survival of E. coli to MEK treatment. At 37° C. there was fullsurvival at 4% w/v and no survival at 6% w/v, while at 28° C. there wasfull survival at 6% w/v. Thus, the tolerance of E. coli to MEK increasedwith decreasing exposure temperature.

Example 4 Increased Tolerance of E. coli and L. plantarum PN0512 to1-Butanol at Decreased Exposure Temperature

This Example demonstrates that the toxic effects of 1-butanol and2-butanol on various microbial cells was reduced at lower temperatures.This was demonstrated by incubating E. coli (strain MG1655; ATCC#700926), and L. plantarum (strain PN0512; ATCC # PTA-7727) with either1-butanol or 2-butanol at different temperatures and then determiningthe fraction of the cells that survived the treatment at the differenttemperatures.

Using overnight cultures or cells from plates, 30 mL cultures of themicroorganisms to be tested were started in the following culture media:

-   -   E. coli—Miller's LB medium (Teknova, Half Moon Bay, Calif.):    -   L. plantarum PN0512—Lactobacilli MRS Broth (BD Diagnostic        Systems, Sparks, Md.).        The E. coli and L. plantarum cultures were grown at 37° C.        aerobically with shaking until the cultures were in log phase        and the OD₆₀₀ was between 0.6 and 0.8. A 50 μL aliquot of each        culture was removed for a time zero sample. The remainder of the        cultures was divided into six 5 mL portions and placed in six        small incubation flasks or tubes. Different amounts of 1-butanol        or 2-butanol were added to the six flasks to bring the        concentration to predetermined values, as listed in the tables        below. The flasks or tubes were incubated at a desired        temperature, aerobically without shaking for 1 h. After the        incubation with one of the butanols, 2 μL from each of the        flasks (and in addition 2 μL of the time zero sample of the        culture before exposure to one of the butanols) were pipetted        into the “head” wells of a 96 well (8×12) microtiter plate, each        containing 198 μL of LB medium to give a 10⁻² dilution of the        culture. Subsequently, 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶ serial        dilutions of the cultures were prepared as follows. The 10⁻³        dilution was prepared by pipetting 20 μL of the sample from the        head well into the 180 μL LB medium in the next well using a        multi-channel pipette. This procedure was repeated 3 more times        on successive wells to prepare the 10⁻⁴, 10⁻⁵, and 10⁻⁶        dilutions. After each liquid transfer, the solution in the well        was mixed by pipetting it up and down 10 times with the        multi-channel pipetor. A 5 μL aliquot of each dilution was        spotted onto an LB plate using a multi-channel pipette starting        with the 10⁻⁶ dilution, then the 10⁻⁵, and so on working from        more to less dilute without a change of tips. The spots were        allowed to soak into the agar by leaving the lid of the plate        slightly open for 15 to 30 min in a sterile transfer hood. The        plates were covered, inverted, and incubated overnight at 37° C.        The following day, the number of colonies in the spots were        counted from the different dilutions. The number of living        cells/mL in each of the original culture solutions from which        the 2 μL was withdrawn was calculated and compared to the number        of cells in the control untreated culture to determine the % of        the cells surviving.

The results of experiments in which E. coli cells were treated with1-butanol at temperatures of 0, 30, and 37° C. are shown Table 12.

TABLE 12 Percentage of E. coli cells surviving in 1-butanol at 0, 30 and37° C. 1-butanol % Survival % v/v % Survival at 0° C. % Survival at 30°C. at 37° C. 0 100 100 100  1 nt¹ 100 72 1.5 nt 100 20 2 nt 100  0 2.5100  23  0 3 100  0  0 3.5 100  0 nt 4 100 nt nt 4.5 100 nt nt ¹“nt” =not tested

The concentration at which 1-butanol kills E. coli cells was affected bythe treatment temperature. At 0° C., concentrations of 1-butanol as highas 4.5% v/v had no toxic effect on E. coli cells during a one hourtreatment. At 30° C., E. coli cells were killed when treated with 3% v/v1-butanol for one hour. At 37° C., E. coli cells were killed whentreated with 2% v/v 1-butanol for one hour.

The results of experiments in which L. plantarum PN0512 cells weretreated with 1-butanol at temperatures of 0, 23, and 37° C. for one hourare shown Table 13.

TABLE 13 Percentage of L. plantarum PN0512 cells surviving in 1-butanolat 0, 23 and 37° C. 1-butanol % Survival % Survival % v/v at 0° C. %Survival at 23° C. at 37° C. 0 100 100 100  1 nt¹ nt 80 1.5 nt nt 58 2nt 100 29 2.5 nt 100  8 3 100  82  0 3.5 100  0  0 4 100  0 nt 4.5 100 0 nt 5  0 nt nt 5.5  0 nt nt ¹“nt” = not tested

The concentration at which 1-butanol kills L. plantarum PN0512 cells wasaffected by the treatment temperature. At 0° C., concentrations of1-butanol as high as 4.5% v/v had no toxic effect on L. plantarum PN0512cells during a one hour treatment. At 23° C., L. plantarum PN0512 cellswere killed when treated with 3.5% v/v 1-butanol for one hour. At 37°C., L. plantarum PN0512 cells were killed when treated with 2.5% v/v1-butanol for one hour.

Example 5 Cloning and Expression of Acetolactate Synthase

The purpose of this Example was to clone and express in E. coli the budBgene that encodes the enzyme acetolactate synthase. The budB gene wasamplified from Klebsiella pneumoniae strain ATCC 25955 genomic DNA usingPCR.

The budB sequence which encodes acetolactate synthase was amplified fromKlebsiella pneumoniae (ATCC 25955) genomic DNA by PCR using the primerpair B1 (SEQ ID NO:15) and B2 (SEQ ID NO:16). Other PCR amplificationreagents (e.g. Kod HiFi DNA Polymerase (Novagen Inc., Madison, Wis.;catalog no. 71805-3)) were supplied in manufacturers' kits and usedaccording to the manufacturer's protocol. Klebsiella pneumoniae genomicDNA was prepared using the Gentra Puregene Puregene kit (Gentra Systems,Inc., Minneapolis, Minn.; catalog number D-5000A). Amplification wascarried out in a DNA Thermocycler GeneAmp 9700 (PE Applied Biosystems,Foster city, CA). The nucleotide sequence of the open reading frame(ORF) and the predicted amino acid sequence of the enzyme are given asSEQ ID NO:3 and SEQ ID NO:4, respectively.

For expression studies the Gateway cloning technology (Invitrogen Corp.,Carlsbad, Calif.) was used. The entry vector pENTR/SD/D-TOPO allowsdirectional cloning and provided a Shine-Dalgarno sequence for the geneof interest. The destination vector pDEST14 used a T7 promoter forexpression of the gene with no tag. The forward primer incorporated fourbases (CACC) immediately adjacent to the translational start codon toallow directional cloning of the budB acetolactate synthase codingregion PCR product into pENTR/SD/D-TOPO (Invitrogen), generating theplasmid pENTRSDD-TOPObudB. The PENTR construct was transformed into E.coli Top10 (Invitrogen) cells and plated according to the manufacturer'srecommendations. Transformants were grown overnight and plasmid DNA wasprepared using the QIAprep Spin Miniprep kit (Qiagen, Valencia, Calif.;catalog no. 27106) according to the manufacturer's recommendations. Tocreate an expression clone, the budB coding region frompENTRSDD-TOPObudB was transferred to the PDEST 14 vector by in vitrorecombination using the LR Clonase mix (Invitrogen, Corp., Carlsbad,Calif.). The resulting vector, pDEST14budB, was transformed intoBL-21-Al cells (Invitrogen Corp.). BL-21-Al cells carry a chromosomalcopy of the T7 RNA polymerase under control of the arabinose-induciblearaBAD promoter.

Transformants are inoculated into LB medium supplemented with 50 μg/mLof ampicillin and grown overnight. An aliquot of the overnight cultureis used to inoculate 50 mL of LB medium supplemented with 50 μg/mL ofampicillin. The culture is incubated at 37° C. with shaking until theOD₆₀₀ reaches 0.6-0.8. The culture is split into two 25-mL portions andarabinose is added to one of the flasks to a final concentration of 0.2%w/v. The negative control flask is not induced with arabinose. Theflasks are incubated for 4 h at 37° C. with shaking. Cells are harvestedby centrifugation and the cell pellets are resuspended in 50 mM MOPS, pH7.0 buffer. The cells are disrupted either by sonication or by passagethrough a French Pressure Cell. Each cell lysate is centrifuged yieldingthe supernatant and the pellet or the insoluble fraction. An aliquot ofeach fraction (whole cell lysate, from induced and control cells, isresuspended in SDS (MES) loading buffer (Invitrogen), heated to 85° C.for 10 min and subjected to SDS-PAGE analysis (NuPAGE 4-12% Bis-TrisGel, catalog no. NP0322Box, Invitrogen). A protein of the expectedmolecular weight, as deduced from the nucleic acid sequence, is presentin the induced culture but not in the uninduced control.

Acetolactate synthase activity in the cell free extracts is measuredusing the method described by Bauerle et al. (Bauerle et al. (1964)Biochim. Biophys. Acta 92:142-149). Protein concentration is measured byeither the Bradford method or by the Bicinchoninic Kit (Sigma, catalogno. BCA-1; St. Louis, Mo.) using Bovine serum albumin (BSA) (Bio-Rad,Hercules, Calif.) as the standard.

Example 6 Cloning and Expression of Acetolactate Decarboxylase

The purpose of this Example was to clone and express in E. coli the budAgene that encodes the enzyme acetolactate decarboxylase. The budA genewas amplified from Klebsiella pneumoniae strain ATCC 25955 genomic DNAusing PCR.

The budA sequence which encodes acetolactate decarboxylase, was clonedin the same manner as described for budB in Example 5, except that theprimers used for PCR amplification were B3 (SEQ ID NO:17) and B4 (SEQ IDNO:18). The nucleotide sequence of the open reading frame (ORF) and thepredicted amino acid sequence of the enzyme are given as SEQ ID NO:1 andSEQ ID NO:2, respectively. The resulting plasmid was namedpENTRSDD-TOPObudA.

Acetolactate decarboxylase activity in the cell free extracts ismeasured using the method described by Bauerle et al., supra.

Example 7 Prophetic Cloning and Expression of Butanediol Dehydrogenase

The purpose of this prophetic Example is to describe how to clone andexpress in E. coli the budC gene that encodes the enzyme butanedioldehydrogenase. The budC gene is amplified from Klebsiella pneumoniaestrain IAM1063 genomic DNA using PCR.

The budC sequence encoding butanediol dehydrogenase is cloned andexpressed in the same manner as described for budA in Example 5, exceptthat the primers used for PCR amplification are B5 (SEQ ID NO:19) and B6(SEQ ID NO:20) and the genomic template DNA is from Klebsiellapneumoniae IAM1063 (which is obtained from the Institute of AppliedMicrobiology Culture Collection, Tokyo, Japan). Klebsiella pneumoniaeIAM1063 genomic DNA is prepared using the Gentra Puregene Puregene kit(Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5000A). Thenucleotide sequence of the open reading frame (ORF) and the predictedamino acid sequence of the enzyme are given as SEQ ID NO:5 and SEQ IDNO:6, respectively.

Butanediol dehydrogenase activity in the cell free extracts is measuredspectrophotometrically by following NADH consumption at an absorbance of340 nm.

Example 8 Prophetic Cloning and Expression of Butanediol Dehydratase

The purpose of this prophetic Example is to describe how to clone andexpress in E. coli the pddA, pddB and pddC genes that encode butanedioldehydratase. The pddA, pddB and pddC genes are amplified from Klebsiellaoxytoca ATCC 8724 genomic DNA using PCR.

The pddA, pddB and pddC sequences which encode butanediol dehydrataseare cloned and expressed in the same manner as described for budA inExample 5, except that the genomic template DNA is from Klebsiellaoxytoca ATCC 8724, and the primers are B7 (SEQ ID NO:21) and B8 (SEQ IDNO:22). Klebsiella oxytoca genomic DNA is prepared using the GentraPuregene Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalognumber D-5000A). A single PCR product including all three open readingframes (ORFs) is cloned, so that all three coding regions are expressedas an operon from a single promoter on the expression plasmid. Thenucleotide sequences of the open reading frames for the three subunitsare given as SEQ ID NOs:7, 9, and 11, respectively, and the predictedamino acid sequences of the three enzyme subunits are given as SEQ IDNOs:8, 10, and 12, respectively.

Butanediol dehydratase activity in the cell free extracts is measured byderivatizing the ketone product with 2,4-dinitrophenylhydrazine (DNPH).Briefly, 100 μL of reaction mixture, cell extract containingapproximately 0.0005 units of enzyme, 40 mM potassium phosphate buffer(pH 8.0), 2 μg of adenosylcobalamin, 5 μg of 2,3,-butanediol, and 1 μgof bovine serum albumin, is quenched by addition of an equal volume of0.05 wt % DNPH in 1.0 N HCl. After 15 min at room temperature, the coloris developed by addition of 100 μL of 4 N NaOH. The amount of product isdetermined from the absorbance of the final solution at 550 nm comparedto a standard curve prepared with 2-butanone. All reactions are carriedout at 37° C. under dim red light.

Example 9 Prophetic Cloning and Expression of Butanol Dehydrogenase

The purpose of this prophetic Example is to describe how to clone andexpress in E. coli the sadh gene that encodes butanol dehydrogenase. Thesadh gene is amplified from Rhodococcus ruber strain 219 genomic DNAusing PCR.

The sadh sequence encoding butanol dehydrogenase is cloned and expressedin the same manner as described for budA in Example 5, except that thegenomic template DNA is from Rhodococcus ruber strain 219 (Meens,Institut fuer Mikrobiologie, Universitaet Hannover, Hannover, Germany)and the primers are B9 (SEQ ID NO:23) and B10 (SEQ ID NO:24).Rhodococcus ruber genomic DNA is prepared using the Ultra Clean™Microbial DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad,Calif.), according to the manufacturer's protocol. The nucleotidesequence of the open reading frame (ORF) and the predicted amino acidsequence of the enzyme are given as SEQ ID NO:13 and SEQ ID NO:14,respectively.

Butanol dehydrogenase activity in cell free extracts is measured byfollowing the increase in absorbance at 340 nm resulting from theconversion of NAD to NADH when the enzyme is incubated with NAD and2-butanol.

Example 10 Prophetic Construction of a Transformation Vector for theGenes in a 2-Butanol Biosynthetic Pathway

The purpose of this prophetic Example is to describe the preparation ofa transformation vector for the genes in a 2-butanol biosyntheticpathway (i.e., Pathway 3 as described above). Like most organisms, E.coli converts glucose initially to pyruvic acid. The enzymes required toconvert pyruvic acid to 2-butanol following Pathway 3, i.e.,acetolactate synthase, acetolactate decarboxylase, butanedioldehydrogenase, butanediol dehydratase, and butanol dehydrogenase, areencoded by the budA, budB, budC, pddA, pddB, pddC and sadh genes. Tosimplify building the 2-butanol biosynthetic pathway in a recombinantorganism, the genes encoding the 5 steps in the pathway are divided intotwo operons. The upper pathway comprises the first three steps catalyzedby acetolactate synthase, acetolactate decarboxylase, and butanedioldehydrogenase. The lower pathway comprises the last two steps catalyzedby butanediol dehydratase and butanol dehydrogenase.

The coding sequences are amplified by PCR with primers that incorporaterestriction sites for later cloning, and the forward primers contain anoptimized E. coli ribosome binding site (AAAGGAGG). PCR products areTOPO cloned into the pCR4Blunt-TOPO vector and transformed into Top10cells (Invitrogen). Plasmid DNA is prepared from the TOPO clones, andthe sequence of the cloned PCR fragment is verified. Restriction enzymesand T4 DNA ligase (New England Biolabs, Beverly, Mass.) are usedaccording to manufacturer's recommendations. For cloning experiments,restriction fragments are gel-purified using QIAquick Gel Extraction kit(Qiagen).

After confirmation of the sequence, the coding regions are subclonedinto a modified pUC19 vector as a cloning platform. The pUC19 vector ismodified by a HindIII/SapI digest, followed by treatment with Klenow DNApolymerase to fill in the ends. The 2.4 kB vector fragment isgel-purified and religated creating pUC19dHS. Alternatively the pUC19vector is modified by a SphI/SapI digest, followed by treatment withKlenow DNA polymerase to blunt the ends. The 2.4 kB vector fragment isgel-purified and religated creating pUC19dSS. The digests remove the lacpromoter adjacent to the MCS (multiple cloning sites), preventingtranscription of the operons from the vector.

Upper Pathway:

The budABC coding regions are amplified from Klebsiella pneumoniaegenomic DNA by PCR using primer pair B11 and B12 (Table 4), given as SEQID NOs:25 and 26, respectively. The forward primer incorporates an EcoRIrestriction site and a ribosome binding site (RBS). The reverse primerincorporates an SphI restriction site. The PCR product is cloned intopCR4Blunt-TOPO creating pCR4Blunt-TOPO-budABC.

To construct the upper pathway operon pCR4Blunt-TOPO-budABC is digestedwith EcoRI and SphI releasing a 3.2 kbp budABC fragment. The pUC19dSSvector is also digested with EcoRI and SphI, releasing a 2.0 kbp vectorfragment. The budABC fragment and the vector fragment are ligatedtogether using T4 DNA ligase (New England Biolabs) to formpUC19dSS-budABC.

Lower Pathway:

The pddABC coding regions are amplified from Klebsiella oxytoca ATCC8724 genomic DNA by PCR using primers B13 and B14 (Table 4), given asSEQ ID NOs:27 and 28, respectively, creating a 2.9 kbp product. Theforward primer incorporates EcoRI and PmeI restriction sites and a RBS.The reverse primer incorporates the BamHI restriction site. The PCRproduct is cloned into pCRBlunt II-TOPO creating pCRBluntII-pdd.

The sadh gene is amplified from Rhodococcus ruber strain 219 genomic DNAby PCR using primers B15 and B16 (Table 4), given as SEQ ID NOs:29 and30, respectively, creating a 1.0 kbp product. The forward primerincorporates a BamHI restriction site and a RBS. The reverse primerincorporates an XbaI restriction site. The PCR product is cloned intopCRBlunt II-TOPO creating pCRBluntII-sadh.

To construct the lower pathway operon, a 2.9 kbp EcoRI and BamHIfragment from pCRBluntII-pdd, a 1.0 kbp BamHI and XbaI fragment frompCRBluntII-sadh, and the large fragment from an EcoRI and XbaI digest ofpUC19dHS are ligated together. The three-way ligation createspUC19dHS-pdd-sadh.

The pUC19dSS-budABC vector is digested with PmeI and HindIII, releasinga 3.2 kbp fragment that is cloned into pBenBP, an E. coli-B. subtilisshuttle vector. Plasmid pBenBP is created by modification of the pBE93vector, which is described by Nagarajan (WO 93/2463, Example 4). Togenerate pBenBP, the Bacillus amyloliquefaciens neutral proteasepromoter (NPR) signal sequence and the phoA gene are removed from pBE93with an NcoI/HindIII digest. The NPR promoter is PCR amplified frompBE93 by primers BenF and BenBPR, given by SEQ ID NOs:31 and 32,respectively. Primer BenBPR incorporates BstEII, PmeI and HindIII sitesdownstream of the promoter. The PCR product is digested with NcoI andHindIII, and the fragment is cloned into the corresponding sites in thevector pBE93 to create pBenBP. The upper operon fragment is subclonedinto the PmeI and HindIII sites in pBenBP creating pBen-budABC.

The pUC19dHS-pdd-sadh vector is digested with PmeI and HindIII releasinga 3.9 kbp fragment that is cloned into the PmeI and HindIII sites ofpBenBP, creating pBen-pdd-sadh.

Example 11 Prophetic Expression of a 2-Butanol Biosynthetic Pathway inE. coli

The purpose of this prophetic Example is to describe how to express a2-butanol biosynthetic pathway in E. coli.

The plasmids pBen-budABC and pBen-pdd-sadh, prepared as described inExample 10, are separately transformed into E. coli NM522 (ATCC No.47000), and expression of the genes in each operon is monitored bySDS-PAGE analysis and enzyme assay. After confirmation of expression ofall genes, pBen-budABC is digested with EcoRI and HindIII to release theNPR promoter-budABC fragment. The fragment is blunt ended using theKlenow fragment of DNA polymerase (New England Biolabs, catalog no.M0210S). The plasmid pBen-pdd-sadh is digested with EcoRI and similarlyblunted to create a linearized, blunt-ended vector fragment. The vectorand NPR-budABC fragments are ligated, creating p2BOH. This plasmid istransformed into E. coli NM522 to give E. coli NM522/p2BOH, andexpression of the genes is monitored as previously described.

E. coli NM522/p2BOH is inoculated into a 250 mL shake flask containing50 mL of medium and shaken at 250 rpm and 35° C. The medium is composedof: dextrose, 5 g/L; MOPS, 0.05 M; ammonium sulfate, 0.01 M; potassiumphosphate, monobasic, 0.005 M; S10 metal mix, 1% (v/v); yeast extract,0.1% (w/v); casamino acids, 0.1% (w/v); thiamine, 0.1 mg/L; proline,0.05 mg/L; and biotin 0.002 mg/L, and is titrated to pH 7.0 with KOH.S10 metal mix contains: MgCl₂, 200 mM; CaCl₂, 70 mM; MnCl₂, 5 mM; FeCl₃,0.1 mM; ZnCl₂, 0.1 mM; thiamine hydrochloride, 0.2 mM; CuSO₄, 172 μM;CoCl₂, 253 μM; and Na₂MoO₄, 242 μM. After 18 h, 2-butanol is detected byHPLC or GC analysis using methods that are well known in the art, forexample, as described in the General Methods section above.

Example 12 Prophetic Expression of a 2-Butanol Biosynthetic Pathway inBacillus subtilis

The purpose of this prophetic Example is to describe how to express a2-butanol biosynthetic pathway in Bacillus subtilis.

The plasmids pBen-budABC and pBen-pdd-sadh, prepared as described inExample 10, are separately transformed into Bacillus subtilis BE1010 (J.Bacteriol. 173:2278-2282 (1991)) and expression of the genes in eachoperon is monitored as described in Example 11. The plasmid pBen-budABCis digested with EcoRI and HindIII to release the NPR promoter-budABCfragment. The fragment is blunt ended using the Klenow fragment of DNApolymerase (New England Biolabs, catalog no. M0210S). The plasmidpBen-pdd-sadh is digested with EcoRI and similarly blunted to create alinearized, blunt-ended vector fragment. The vector and NPR-budABCfragments are ligated, creating p2BOH. This plasmid is transformed intoBacillus subtilis BE1010 to give Bacillus subtilis BE1010/p2BOH, andexpression of the genes is monitored as previously described.

Bacillus subtilis BE1010/p2BOH is inoculated into a 250 mL shake flaskcontaining 50 mL of medium and shaken at 250 rpm and 35° C. for 18 h.The medium is composed of: dextrose, 5 g/L; MOPS, 0.05 M; glutamic acid,0.02 M; ammonium sulfate, 0.01 M; potassium phosphate, monobasic buffer,0.005 M; S10 metal mix (as described in Example 11), 1% (v/v); yeastextract, 0.1% (w/v); casamino acids, 0.1% (w/v); tryptophan, 50 mg/L;methionine, 50 mg/L; and lysine, 50 mg/L, and is titrated to pH 7.0 withKOH. After 18 h, 2-butanol is detected by HPLC or GC analysis usingmethods that are well known in the art, for example, as described in theGeneral Methods section above.

Example 13 Construction of a Transformation Vector for the Genes in a2-Butanol Biosynthetic Pathway

The purpose of this Example was to prepare a recombinant E. coli hostcarrying the genes in a 2-butanol biosynthetic pathway (i.e., Pathway 3as described above). Like most organisms, E. coli converts glucoseinitially to pyruvic acid. The enzymes required to convert pyruvic acidto 2-butanone in Pathway 3, i.e., acetolactate synthase, acetolactatedecarboxylase, butanediol dehydrogenase, and butanediol dehydratase areencoded by the budA, budB, budC, pddA, pddB, and pddC genes. In the laststep of the pathway, a butanol dehydrogenase converts 2-butanone to2-butanol. Dehydrogenases that carry out this last step are promiscuousand may be found in many organisms. To simplify building the 2-butanolbiosynthetic pathway in a recombinant organism, the genes encoding the 5steps in the pathway were divided into multiple operons. The upperpathway operon comprised the first three steps catalyzed by acetolactatesynthase, acetolactate decarboxylase, and butanediol dehydrogenase andwere cloned onto an expression vector. The lower pathway comprised thelast two steps catalyzed by butanediol dehydratase including thereactivating factor (Mori et al., J. Biol. Chem. 272:32034 (1997)) and abutanol dehydrogenase. The diol dehydratase can undergo suicideinactivation during catalysis. The reactivating factor protein encodedby ddrA and ddrB (GenBank AF017781, SEQ ID NO:70) reactivates theinactive enzyme. The ddrA and ddrB genes flank the diol dehydrataseoperon. The operons for the dehydratase/reactivating factor and thebutanol dehydrogenase were either cloned onto another expression vectoror the dehydratase/reactivating factor operon was cloned singly ontoanother expression vector and the last step was provided by anendogenous activity in the demonstration host.

Construction of Vector pTrc99a-budABC:

The budAB coding regions were amplified from K. pneumoniae ATCC 25955genomic DNA by PCR using primer pair BABC F and BAB R, given as SEQ IDNOs:33 and 34, respectively (see Table 4), creating a 2.5 kbp product.The forward primer incorporated SacI and EcoRI restriction sites and aribosome binding site (RBS). The reverse primer incorporated a SpeIrestriction site. The PCR product was cloned into pCR4Blunt-TOPOcreating pCR4Blunt-TOPO-budAB. Plasmid DNA was prepared from the TOPOclones and the sequence of the genes was verified with primers M13Forward (SEQ ID NO:35), M13 Reverse (SEQ ID NO:36), N83 SeqF2 (SEQ IDNO:37), N83 SeqF3 (SEQ ID NO:38) and N84 SeqR4 (SEQ ID NO:39) (see Table5).

The budC coding region was amplified from K. pneumoniae ATCC 25955genomic DNA by PCR using primer pair BC Spe F and BC Xba R given as SEQID NOs:40 and 41, respectively, creating a 0.8 kbp product. The forwardprimer incorporated a SpeI restriction site, a RBS and modified the CDSby changing the second and third codons from AAA to AAG. The reverseprimer incorporated an XbaI restriction site. The PCR product was clonedinto pCR4 Blunt-TOPO creating pCR4 Blunt-TOPO-budC. Plasmid DNA wasprepared from the TOPO clones and the sequence of the genes was verifiedwith primers M13 Forward (SEQ ID NO:35) and M13 Reverse (SEQ ID NO:36).

To construct the budABC operon, pCR4 Blunt-TOPO-budC was digested withSnaBI and XbaI releasing a 1.0 kbp budC fragment. The vector pTrc99a(Amann et al., Gene 69(2):301-315 (1988)) was digested with SmaI andXbaI creating a 4.2 kbp linearized vector fragment. The vector and thebudC fragment were ligated to create pTrc99a-budC and transformed intoE. coli Top 10 cells (Invitrogen). Transformants were analyzed by PCRamplification with primers Trc F (SEQ ID NO:42) and Trc R (SEQ ID NO:43)for a 1.2 kbp product to confirm the presence of the budC insert. ThebudAB genes were subcloned from pCR4 Blunt-TOPO-budAB as a 2.5 kbpEcoRI/SpeI fragment. Vector pTrc99a-budC was digested with EcoRI andSpeI and the resulting 5.0 kbp vector fragment was gel-purified. Thepurified vector and budAB insert were ligated and transformed into E.coli Top 10 cells. Transformants were screened by PCR amplification withprimers Trc F (SEQ ID NO:42) and N84 Seq R2 (SEQ ID NO:65) to confirmcreation of pTrc99a-budABC. In this plasmid, the bud A, B, and C codingregions are adjacent to each other, in this order, and between the Trcpromoter and the rrnB termination sequence.

Results:

Three independent isolates of E. coli Top 10/pTrc99a-budABC wereexamined for the production of butanediol, using E. coli Top10/pCL1925-Kodd-ddr (described below) as a negative control. The strainswere grown in LB medium containing 100 μg/mL carbenicillin. Theresulting cells were used to inoculate shake flasks (approximately 175mL total volume) containing 125 mL of TM3a/glucose medium with 100 μg/mLcarbenicillin. In addition, the flasks inoculated with strains carryingpTrc99a-budABC contained 0.4 mM isopropyl β-D-1-thiogalactopyranoside(IPTG). TM3a/glucose medium contains (per liter): 10 g glucose, 13.6 gKH₂PO₄, 2.0 g citric acid monohydrate, 3.0 g (NH₄)₂SO₄, 2.0 gMgSO₄.7H₂O, 0.2 g CaCl₂.2H₂O, 0.33 g ferric ammonium citrate, 1.0 mgthiamine HCl, 0.50 g yeast extract, and 10 mL trace elements solution,adjusted to pH 6.8 with NH₄OH. The solution of trace elements contained:citric acid H₂O (4.0 g/L), MnSO₄.H₂O (3.0 g/L), NaCl (1.0 g/L),FeSO₄.7H₂O (0.10 g/L), CoCl₂.6H₂O (0.10 g/L), ZnSO₄.7H₂O (0.10 g/L),CuSO₄.5H₂O (0.010 g/L), H₃BO₃ (0.010 g/L), and Na₂MoO₄ 2H₂O (0.010 g/L).The flasks, capped with vented caps, were inoculated at a starting OD₆₀₀of approximately 0.03 units and incubated at 34° C. with shaking at 300rpm.

Approximately 23 h after induction, an aliquot of the broth was analyzedby HPLC (Shodex Sugar SH1011 column) and GC(HP-INNOWax), using the samemethods described in the General Methods section for 2-butanol and2-butanone. The results of the analysis are given in Table 14. The threeE. coli clones converted glucose to acetoin and meso-2,3-butanediol, thedesired intermediates of the pathway, with a molar selectivity of 14%.This selectivity was approximately 35-fold higher than that observedwith the E. coli control strain lacking budABC.

TABLE 14 Production of Acetoin and meso-2,3-butanediol by E. coli Top10/pTrc99a-budABC Meso-2,3- Butanediol, Molar Strain OD₆₀₀ Acetoin, mMmM Selectivity^(a), % Negative 1.4 0.07 0.03 0.4 control Isolate #1 1.50.64 1.3 14 Isolate #2 1.4 0.70 1.2 14 Isolate #3 1.4 0.74 1.3 15^(a)Molar selectivity is (acetoin + meso-2,3-butanendiol)/(glucoseconsumed).Construction of Vector pCL1925-KoDD-ddr:

The diol dehydratase (GenBank D45071, SEQ ID NO:69) and reactivatingfactor (GenBank AF017781, SEQ ID NO:70) operons were PCR amplified fromKlebsiella oxytoca ATCC 8724 as a single unit with primers DDo For (SEQID NO: 44) and DDo Rev (SEQ ID NO:45). The forward primer incorporatedan optimized E. coli RBS and a HindIII restriction site. The reverseprimer included an XbaI restriction site. The 5318 bp PCR product wascloned into pCR4Blunt-TOPO and clones of the resultingpCR4Blunt-TOPO-Kodd-ddr were sequenced with primers M13 Forward (SEQ IDNO:35), M13 Reverse (SEQ ID NO:36), DDko seq F2 (SEQ ID NO:46), DDko seqF5 (SEQ ID NO:47), DDko seq F7 (SEQ ID NO:48), DDko seq F9 (SEQ IDNO:49), DDko seq R1 (SEQ ID NO:50), DDko seq R3 (SEQ ID NO:51), DDko seqR7 (SEQ ID NO:52), and DDko seq R10 (SEQ ID NO:53). A clone having theinsert with the expected sequence was identified.

For expression, the diol dehydratase/reactivating factor genes weresubcloned into pCL1925 (U.S. Pat. No. 7,074,608), a low copy plasmidcarrying the glucose isomerase promoter from Streptomcyes.pCR4Blunt-TOPO-Kodd-ddr was digested with HindIII and XbaI and theresulting 5.3 kbp Kodd-ddr fragment was gel-purified. Vector pCL1925 wasdigested with HindIII and XbaI and the resulting 4539 bp vector fragmentwas gel purified. The vector and Kodd-ddr fragment were ligated andtransformed into E. coli Top10. Transformants were screened by PCR withprimers DDko Seq F7 (SEQ ID NO:48) and DDko Seq R7 (SEQ ID NO: 52).Amplification of the plasmid (pCL1925-Kodd-ddr) carrying the insertresulted in a product of approximately 797 bp.

Activity of diol dehydratase towards meso-2,3-butanediol was measured byincubating cell extract (total protein ˜0.8 mg/mL) with 10 mM butanedioland 12 mM coenzyme B₁₂ in 80 mM HEPES (pH 8.2) for 17 h at roomtemperature. Formation of the expected product, 2-butanone, wasdetermined by HPLC as described in the General Methods.

Construction of Vector pCL1925-KoDD-ddr::T5 chnA ter:

To provide a heterologous alcohol dehydrogenase activity, the chnA geneencoding cyclohexanol dehydrogenase from Acinetobacter sp. (Cheng etal., J. Bacteriol. 182:4744-4751 (2000)) was cloned into the pCL1925vector with the diol dehydratase operon, pCL1925-Kodd-ddr. The chnAgene, given as SEQ ID NO:71 (Genbank No: AF282240, SEQ ID NO:73) wasamplified from pDCQ2, a cosmid carrying the cyclohexanol gene clusterfrom Acinetobacter, with primers ChnA F (SEQ ID NO:54) and ChnA R (SEQID NO:55). The resulting 828 bp PCR product was cloned intopCR4Blunt-TOPO to create pCR4Blunt-TOPO-chnA and transformants werescreened by colony PCR with primers M13 Forward (SEQ ID NO:35) and M13Reverse (SEQ ID NO:36). Correct clones produced a PCR product of about 1kbp and were sequenced with primers M13 Forward (SEQ ID NO:35) and M13Reverse (SEQ ID NO:36).

After sequencing pCR4Blunt-TOPO-chnA to confirm the correct sequence,the chnA gene was subcloned from the plasmid as an 813 bp MfeI/SmaIfragment. The expression vector pQE30 (Qiagen) was digested with MfeIand SmaI and the resulting 3350 bp vector fragment was gel-purified. ThechnA fragment and the purified vector were ligated and transformed intoE. coli Top10 cells. Transformants were colony PCR screened with primerschnSeq F1 (SEQ ID NO:56) and chnseq R1 (SEQ ID NO:57) for a 494 bp PCRproduct. This cloning placed the chnA gene under the control of the T5promoter in the plasmid, pQE30-chnA.

To prepare the pCL1925 vector to carry two operons, terminators wereadded to the vector. A tonB terminator-mcs-trpA terminator fragment wasprepared by oligonucleotide annealing with primers Top ter F1 (SEQ IDNO:58), Top ter F2 (SEQ ID NO:59), Bot ter R1 (SEQ ID NO:60) and Bot terR2 (SEQ ID NO:61). The annealed DNA was gel-purified on a 6% PAGE gel(Embi-tec, San Diego, Calif.). Vector pCL1925 was digested with SacI andXbaI and gel-purified. The annealed DNA and vector fragment were ligatedto create pCL1925-ter. Transformants were screened by colony PCRamplification with primers pCL1925 vec F (SEQ ID NO:62) and pCL1925 vecR1 (SEQ ID NO:63) for the presence of a PCR product of approximately 400bp. Positive clones from the PCR screen were sequenced with the sameprimers.

Vector pCL1925-ter was digested with XhoI and PmeI and the resulting4622 bp fragment was gel-purified. pQE30-chnA was digested with NcoI andthe DNA was treated with Klenow DNA polymerase to blunt the ends.pQE30-chnA was then digested with XhoI and the resulting 1.2 kbp T5promoter-chnA fragment was gel-purified. The pCL1925-ter vector and thechnA operon fragment were ligated together to give pCL1925-ter-T5chnAand transformed into E. coli Top10. Transformants were screened bycolony PCR amplification with primers pCL1925 vec F (SEQ ID NO:64) andchnseq R1 (SEQ ID NO:59) for a product of approximately 1 kbp.

To finish building the pathway vector, the pCL1925-KoDD-ddr plasmid wasdigested with XbaI and SacI and the resulting 9504 bp vector fragmentwas gel-purified. The chnA operon flanked by terminators, with the trpAterminator (Koichi et al. (1997) Volume 272, Number 51, pp. 32034-32041)3′ to the chnA coding sequence, from pCL1925-ter-T5chnA was gel-purifiedas a 1271 bp XbaI/SacI fragment. After ligation of the fragments andtransformation into E. coli Top10, transformants were screened by colonyPCR. Primers chnseq F1 (SEQ ID NO:58) and pCL1925 vec R2 (SEQ ID NO:64)amplified the expected 1107 bp PCR product in the resulting plasmid,pCL1925-KoDD-ddr::ter-T5chnA.

Example 14 Expression of a 2-Butanol Biosynthetic Pathway in E. coliwith Overexpressed Endogenous Alcohol Dehydrogenase

The purpose of this Example was to express a 2-butanol biosyntheticpathway in several E. coli strains.

Construction of E. coli Strains Constitutively Expressing yqhD:

E. coli contains a native gene (yqhD) that was identified as a1,3-propanediol dehydrogenase (U.S. Pat. No. 6,514,733). The yqhD gene,given as SEQ ID NO:74, has 40% identity to the gene adhB in Clostridium,a probable NADH-dependent butanol dehydrogenase. The yqhD gene wasplaced under the constitutive expression of a variant of the glucoseisomerase promoter 1.6GI (SEQ ID NO:67) in E. coli strain MG16551.6yqhD::Cm (WO 2004/033646) using λ Red technology (Datsenko andWanner, Proc. Natl. Acad. Sci. U.S.A. 97:6640 (2000)). Similarly, thenative promoter was replaced by the 1.5GI promoter (WO 2003/089621) (SEQID NO:68), creating strain MG1655 1.5yqhD::Cm, thus, replacing the 1.6GIpromoter of MG1655 1.6yqhD::Cm with the 1.5GI promoter. The 1.5GI and1.6GI promoters differ by 1 bp in the −35 region, thereby altering thestrength of the promoters (WO 2004/033646). While replacing the nativeyqhD promoter with either the 1.5GI or 1.6GI promoter, the yqhC geneencoding the putative transcriptional regulator for the yqh operon wasdeleted. Butanol dehydrogenase activity was confirmed by enzyme assayusing methods that are well known in the art.

Transformation of E. coli Strains:

Pathway plasmids pCL1925-Kodd-ddr and pTrc99a-budABC, described inExample 13, were co-transformed into E. coli strains MG1655, MG16551.6yqhD, and MG1655 1.5yqhD. The two latter strains overexpress the1,3-propanediol dehydrogenase, YqhD, which also has butanoldehydrogenase activity. Strains were examined for the production of2-butanone and 2-butanol essentially as described above. Cells wereinoculated into shake flasks (approximately 175 mL total volume)containing either 50 or 150 mL of TM3a/glucose medium (with 0.1 mg/Lvitamin B₁₂, appropriate antibiotics and IPTG) to represent medium andlow oxygen conditions, respectively. Spectinomycin (50 μg/mL) andcarbenicillin (100 μg/mL) were used for plasmids pCL1925-Kodd-ddr andpTrc99a-budABC, respectively. The flasks were inoculated at a startingOD₆₀₀ of ≦0.04 units and incubated at 34° C. with shaking at 300 rpm.The flasks containing 50 mL of medium were capped with vented caps; theflasks containing 150 mL, were capped with non-vented caps to minimizeair exchange. IPTG was present at time zero at a concentration of zeroor 0.04 mM. Analytical results for 2-butanone and 2-butanol productionare presented in Table 15. All the E. coli strains comprising a2-butanol biosynthetic pathway produced 2-butanone under low and mediumoxygen conditions and produced 2-butanol under low oxygen conditions.

TABLE 15 Production of 2-Butanone and 2-Butanol by E. coli MG1655strains harboring pathway plasmids pCL1925-Kodd-ddr and pTrc99a-budABCVolume of 2-Butanone, 2-Butanol, Strain^(a,b) IPTG, mM Medium, mL mM mMMG1655 #1 0 50 0.08 Not detected MG1655 #2 0 50 0.11 Not detected MG1655#1 0.04 50 0.12 Not detected MG1655 #2 0.04 50 0.11 Not detected MG1655#1 0 150 0.15 0.047 MG1655 #2 0 150 0.19 0.041 MG1655 #1 0.04 150 0.100.015 MG1655 #2 0.04 150 0.11 0.015 MG1655 0 50 0.10 Not detected1.5yqhD #1 MG1655 0 50 0.07 Not detected 1.5yqhD #2 MG1655 0.04 50 0.12Not detected 1.5yqhD #1 MG1655 0.04 50 0.18 Not detected 1.5yqhD #2MG1655 0 150 0.16 0.030 1.5yqhD #1 MG1655 0 150 0.18 0.038 1.5yqhD #2MG1655 0.04 150 0.10 0.021 1.5yqhD #1 MG1655 0.04 150 0.09 0.017 1.5yqhD#2 MG1655 0 50 0.08 Not detected 1.6yqhD #1 MG1655 0 50 0.07 Notdetected 1.6yqhD #2 MG1655 0.04 50 0.12 Not detected 1.6yqhD #1 MG16550.04 50 0.15 Not detected 1.6yqhD #2 MG1655 0 150 0.17 0.019 1.6yqhD #1MG1655 0 150 0.18 0.041 1.6yqhD #2 MG1655 0.04 150 0.11 0.026 1.6yqhD #1MG1655 0.04 150 0.11 0.038 1.6yqhD #2 Control Not detected Not detected(uninoculated medium) ^(a)#1 and #2 represent independent isolates.^(b)MG1655 is MG1655/pCL1925-Kodd-ddr/pTrc99a-budABC MG1655 1.6yqhD isMG1655 1.6yqhD/pCL1925-Kodd-ddr/pTrc99a-budABC MG1655 1.6yqhD is MG16551.5yqhD/pCL1925-Kodd-ddr/pTrc99a-budABC.

Example 15 Expression of a 2-Butanol Biosynthetic Pathway in E. coliwith Heterologous Alcohol Dehydrogenase

Plasmids pCL1925-KoDD-ddr::ter-T5chnA and pTrc99a-budABC, described inExample 13, were transformed into E. coli strains MG1655 and MG1655ΔyqhCD for a demonstration of the production of 2-butanol.

MG1655 ΔyqhCD carries a yqhCD inactivation that was made using themethod of Datsenko and Wanner (Proc. Natl. Acad. Sci. U.S.A.97(12):6640-6645 (2000)). After replacement of the region with theFRT-CmR-FRT cassette of pKD3, the chloramphenicol resistance marker wasremoved using the FLP recombinase. The sequence of the deleted region isgiven as SEQ ID NO:66.

Strains MG1655/pTrc99a-budABC/pCL1925KoDD-ddr::ter-T5 chnA and MG1655ΔyqhCD/pTrc99a-budABC/pCL1925KoDD-ddr::ter-T5 chnA were examined for theproduction of 2-butanone and 2-butanol essentially as described above.Strain MG1655 ΔyqhCD/pCL1925 was used as a negative control. Cells wereinoculated into shake flasks (approximately 175 mL total volume)containing 50 or 150 mL of TM3a/glucose medium (with 0.1 mg/L vitaminB₁₂ and appropriate antibiotics) to represent medium and low oxygenconditions, respectively. Spectinomycin (50 μg/mL) and ampicillin (100μg/mL) were used for selection of pCL1925 based plasmids andpTrc99a-budABC, respectively. Enzyme activity derived frompTrc99a-budABC was detected by enzyme assay in the absence of IPTGinducer, thus, IPTG was not added to the medium. The flasks wereinoculated at a starting OD₆₀₀ of ≦0.01 units and incubated at 34° C.with shaking at 300 rpm for 24 h. The flasks containing 50 mL of mediumwere capped with vented caps; the flasks containing 150 mL, were cappedwith non-vented caps to minimize air exchange. Analytical results for2-butanone and 2-butanol production are presented in Table 16. Both E.coli strains comprising a 2-butanol biosynthetic pathway produced2-butanone under low and medium oxygen conditions and produced 2-butanolunder low oxygen conditions, while the negative control strain did notproduce detectable levels of either 2-butanone or 2-butanol.

TABLE 16 Production of 2-butanone and 2-butanol by E. coli strainsStrain^(a) Volume, mL 2-Butanone, mM 2-Butanol, mM Negative control,MG1655 50 Not detected Not detected ΔyqhCD/pCL1925 MG1655/pTrc99a- 500.33 Not detected budABC/pCL1925KoDD-ddr::T5 chnA ter MG1655ΔyqhCD/pTrc99a- 50 0.23 Not detected budABC/pCL1925KoDD-ddr::T5 chnA ter#1 MG1655 ΔyqhCD/pTrc99a- 50 0.19 Not detectedbudABC/pCL1925KoDD-ddr::T5 chnA #2 Negative control, MG1655 150 Notdetected Not detected ΔyqhCD/pCL1925 MG1655/pTrc99a- 150 0.41 0.12budABC/pCL1925KoDD-ddr::T5 chnA ter MG1655 ΔyqhCD/pTrc99a- 150 0.15 0.46budABC/pCL1925KoDD-ddr::T5 chnA #1 MG1655 ΔyqhCD/pTrc99a- 150 0.44 0.14budABC/pCL1925KoDD-ddr::T5 chnA #2 Medium Not detected Not detected^(a)#1 and #2 represent independent isolates.

Example 16 Cloning of Amino:Pvruvate Transaminase (APT)

An amino:pyruvate transaminase (APT) from Vibrio fluvialis JS17 wasidentified by Shin et al. (Appl. Microbiol. Biotechnol. (2003)61:463-471). The amino acid sequence (SEQ ID NO:122) was found to havesignificant homology with ω-amino acid:pyruvate transaminases (Shin andKim (J. Org. Chem. 67:2848-2853 (2002)). It was shown that the Vibriofluvialis APT has transaminase activity towards acetoin.

For expression of the APT enzyme in E. coli, a codon optimized APTcoding region (SEQ ID NO:144) was designed using the preferred E. colicodons with additional considerations such as codon balance and mRNAstability, and synthesized (by DNA2.0; Redwood City, Calif.). The codingregion DNA fragment was subcloned into the pBAD.HisB vector (Invitrogen)between the NcoI and HindIII sites and the resulting plasmid, hereafterreferred to as pBAD.APT1, was transformed into TOP10 cells.

Example 17 Characterization of Vibrio fluvialis APT Alanine:AcetoinAminotransferase Activity

A 5 mL volume of LB broth+100 μg/mL ampicillin was inoculated with afresh colony of TOP10/pBAD:APT1 cells. The culture was incubated at 37°C. for approximately 16 h with shaking (225 rpm). A 300 μL aliquot ofthis culture was used to inoculate 300 mL of the same medium, which wasincubated at 37° C. with shaking (225 rpm). When the culture reached anOD₆₀₀ of 0.8, L-arabinose was added to a final concentration of 0.2%(w/v). The culture was incubated for an additional 16 h, then harvested.The cells were washed once with 100 mM potassium phosphate buffer (pH7.8) and then frozen and stored at −80° C.

To isolate the enzyme, the cell pellet was thawed and resuspended in 8mL of 100 mM potassium phosphate buffer (pH 7) containing 0.2 mMethylenediaminetetraacetate, 1 mM dithiothreitol and 1 tablet ofprotease inhibitor cocktail (Roche; Indianapolis, Ind.). The cells werelysed by two passes through a French pressure cell at 900 psi, and theresulting lysate was clarified by centrifugation for 30 min at 17000×g.Ammonium sulfate was added to 35% saturation, and the solution wasstirred for 30 min at room temperature, at which point precipitatedsolids were removed by centrifugation (30 min, 17000×g). Additionalammonium sulfate was added to the supernatant to give 55% saturation,and the solution was again stirred for 30 min at room temperature. Theprecipitated solids were removed by centrifugation (30 min, 17000×g) andthen resuspended in 5 mL of 100 mM potassium phosphate buffer (pH 7)containing 10 μM pyridoxal 5′-phosphate and 1 mM dithiothreitol. Thissolution was desalted by passage through a PD10 column equilibrated withBuffer A (50 mM bis-tris propane buffer (pH 6) containing 10 μMpyridoxal 5′-phosphate and 1 mM dithiothreitol). The desalted extractwas then loaded onto a 20 mL Q-Fast Flow column pre-equilibrated withBuffer A. APT was eluted with a linear gradient of 0-0.1 M NaCl inBuffer A. The enzyme was detected in eluted fractions by the presence ofa protein band of size ˜50 kD when analyzed by SDS-polyacrylamide gelelectrophoresis and by the characteristic absorbance at 418 nm.Fractions containing the enzyme eluted at ˜0.3 M NaCl. These fractionswere pooled to yield a total of 6 mL of a 5.45 mg/mL solution of enzyme,which was >90% pure, as judged by SDS-polyacrylamide gelelectrophoresis.

The alanine:acetoin aminotransferase activity of APT was assayed using alactic dehydrogenase coupled assay. Reaction mixtures contained 100 mMbis-tris propane (pH 9.0), 10 μM pyridoxal 5′-phosphate, 0-50 mMacetoin, 0-5 mM L-alanine, 0.14 or 0.28 mg/mL purified enzyme, 200 μMNADH and 20 U/mL lactic dehydrogenase (Sigma; St. Louis, Mo.). Thereaction was followed by measuring the change in absorbance at 340 nm,indicative of the oxidation of NADH. Under these conditions, thek_(cat)/K_(m) for acetoin was 10 M⁻¹s⁻¹ and that for L-alanine was 400M⁻¹s⁻¹.

The identity of the expected product 3-amino-2-butanol was confirmed bycomparison to a synthetic standard. A mixture of (R,R)- and(S,S)-3-amino-2-butanol was synthesized by the method of Dickey et al.[J Amer Chem Soc 74:944 (1952)]: 5 g of trans-2,3-epoxybutane wereslowly stirred into 150 mL of cold (4° C.) NH₄OH. The reaction wasslowly warmed to room temperature, sealed and stirred at roomtemperature for an additional 10 days. At this time, excess ammonia andwater and residual epoxybutane were removed by rotary evaporation undervacuum at 40° C. The resulting clear oil (2.9 g) was resuspended inwater to a concentration of 10% (w/v). Production of the desired productwas confirmed by NMR analysis and comparison of the spectrum to thatreported by Levy et al. [Org. Magnetic Resonance 14:214 (1980)]. Amixture of the corresponding (2R,3S)- and (2S,3R)-isomers was producedusing the identical method with the exception that the starting materialwas the cis-isomer of 2,3-epoxybutane.

An analytical method for detection of 3-amino-2-butanol was developedbased on the o-phthaldialdehyde derivatization method for amino aciddetermination reported by Roth [Anal. Chem. 43:880 (1971)]. A 200 μLaliquot of 1 mM 3-amino-2-butanol (mixture of isomers) was mixed with200 μL of a 50 mM solution of borate (pH 9.5), to which was added 10 μLof 5 μL/mL 2-mercaptoethanol in ethanol and 10 μL of 10 mg/mLo-phthaldialdehdye in ethanol. The solution was incubated at roomtemperature for 10 min, at which time the derivative was extracted into200 μL hexane. The hexane was separated from the aqueous solution bydecanting, and 10 μL were injected onto a Chiracel OD HPLC column(Daicel Chemical Industries; Fort Lee, N.J.). The column was runisocratically with a mobile phase of 90:10 hexane:isopropanol at a rateof 1 mL/min. The derivatized isomers of 3-amino-2-butanol were detectedby absorbance at 340 nm with retention times of approximately 15.7 and16.8 min [(2S,3S) and (2R,3R)], and 18.4 and 21.9 min [(2R,3S) and(2S,3R)]. To differentiate the enantiomers in the first mixture, thepure (2R,3R) isomer (Bridge Organics; Vicksburg, Mich.) was also rununder the identical conditions and found to be the 16.8 min peak. Todifferentiate the enantiomers in the second mixture, the mixture wasfirst kinetically resolved using the alanine:acetoin aminotransferase:0.28 mg of purified enzyme was incubated with 10 mM pyruvate and 10 mM3-amino-2-butanol [1:1 mixture of (2R,3S) and (2S,3R) isomers] in 1 mLof 100 mM bis-tris propane (pH 9.0). After 24 h at room temperature, analiquot was removed and analyzed as described above. Analysis revealedthat the 18.4 min peak was 95% depleted, while the 21.9 min peakwas >90% retained. A 100 μL aliquot of the remaining reaction mixturewas mixed with 50 μL of 20 mM NADH and 10 μL of extract from theTOP10/pTrc99a-BudC strain described in Example 13. The BudC enzyme isknown to reduce (R)-acetoin to meso-2,3-butanediol and (S)-acetoin to(S,S)-2,3-butanediol [Ui et al. (2004) Letters in Applied Microbiology39:533-537]. After 3 h, samples were taken from the reaction andanalyzed as described above for acetoin and butanediol. The analysisindicated that the primary product of the reduction wasmeso-2,3-butanediol, indicating that the product of the aminotransferasereaction was (R)-acetoin, and therefore the consumed 3-amino-2-butanolisomer was the (2R,3S) isomer. Thus the retention time of 18.4 min canbe assigned to this isomer and 21.9 to the (2S,3R) isomer.

To confirm that the product of the APT-catalyzed alanine:acetoinaminotransferase reaction was 3-amino-2-butanol, 0.28 mg of purifiedenzyme was incubated with 10 mM acetoin, 10 mM L-alanine, 50 U lacticdehydrogenase and 200 μM NADH in 1 mL of 100 mM bis-tris propane (pH9.0). The reaction mixture was incubated at room temperature for 20 h,after which a 200 μL aliquot was removed and derivatized as describedabove. The retention times of the derivatized products were 15.8 min(major product) and 18.5 min (minor product), matching that of the(2S,3S)- and (2R,3S)-3-amino-2-butanol standards.

Example 18 Identification and Cloning of Erwinia carotovora subsp.atroseptica Amino Alcohol Kinase and Amino Alcohol O-Phosphate Lyase

The purpose of this example is to describe the identification andcloning of sequences encoding an amino alcohol kinase and amino alcoholO-phosphate lyase from the bacterium Erwinia carotovora. These twoenzymes are part of Pathway 1 for the conversion of 3-amino-2-butanol to2-butanone via the intermediate 3-amino-2-butanol phosphate as shown inFIG. 1.

Prediction of the Erwinia Amino Alcohol Kinase and the Amino AlcoholO-Phosphate Lyase

ATP-dependent amino alcohol kinase and amino alcohol O-phosphate lyaseactivities have been detected in several Pseudomonas and Erwiniaspecies, including Pseudomonas sp. P6 (NCIB10431), Pseudomonas putidaNCIB 10558 (Jones et al. (1973) Biochem. J. 134:167-182), Erwiniacarotovora, Erwinia amanas, Erwina milletiae, and Erwinia atroseptica(Jones et al. (1973) Biochem. J. 134:959-968). In these studies, theextracts of the above species were shown to have activity for theenzymatic conversion of aminopropanol through aminopropanol O-phosphateto propionaldehyde, and the conversion of ethanolamine throughethanolamine O-phosphate to acetaldehyde.

The genomic sequence of the Erwinia atroseptica strain in which theseactivities were reported to exist (now designated as Erwinia carotovorasubsp. atroseptica strain SCR11043 (ATCC BAA-672)) has been determinedat the Sanger Institute (Bell et al. Proc. Natl. Acad. Sci. USA 101(30): 11105-11110). Analysis of the putative kinases in the Erwiniacarotovora subsp. atroseptica genome revealed an operon sequence (SEQ IDNO:154) encoding a putative protein (ECA2059; SEQ ID NO:124) that is 39%identical to a Rhizobium loti homoserine kinase and a putative class-IIIpyridoxal phosphate (PLP)-dependent aminotransferase (ECA2060; SEQ IDNO:126) that is 58% identical to a putative aminotransferase fromRhizobium meliloti. We predicted that ECA2059 was an amino alcoholkinase and ECA2060 was an amino alcohol O-phosphate lyase which uses PLPas cofactor.

Cloning of the Putative Amino Alcohol Kinase and Putative Amino AlcoholO-phosphase Lyase from Erwinia carotovora subsp. atroseptica

Genomic DNA of Erwinia carotovora subsp. atroseptica (ATCC #: BAA-672D)was obtained from American Type Culture Collection (ATCC). The operonencoding the putative amino alcohol kinase (KA) and amino alcoholO-phosphate lyase (AT) was named KA-AT (SEQ ID NO:154. This operon wasamplified from the Erwinia genomic DNA by Phusion DNA polymerase(Finnzymes; via New England Biolabs; Ipswich, Mass.) using primers OT872(SEQ. ID. No. 127) and OT873 (SEQ. ID. No128). A DNA fragment of 2.4 kbwas obtained by the PCR reaction, which corresponds to the size of theKA-AT operon. The PCR product was digested with EcoRI and PstIrestriction enzymes, and cloned into vector pKK223-3 (AmershamBiosciences; Piscataway, N.J.) which was digested with the samerestriction enzymes. This produced plasmid pKK223.KA-AT, which containedthe putative Erwinia amino alcohol kinase-lyase operon under control ofthe tac promoter. Similarly, plasmids pKK223.KA and pKK223.AT were madewhich placed the putative Erwinia kinase and the putative Erwinia lyasecoding regions in separate vectors, each under the control of the tacpromoter. For the PCR cloning of the KA coding region (SEQ ID NO:123),primers OT872 (SEQ. ID. No. 127) and OT879 (SEQ. ID. No. 129) were used;and for the PCR cloning of AT coding region (SEQ ID NO:125), primersOT873 (SEQ. ID. No. 128) and OT880 (SEQ. ID. No. 130) were used in thePCR amplifications, which generated PCR products of 1.1 kb and 1.3 kbrespectively. The PCR products were each digested with EcoRI and PstI,and ligated into vector pKK223-3 to generate pKK223.KA and pKK223.AT.

In Vivo Activity of the Putative Amino Alcohol Kinase and Putative AminoAlcohol O-phosphate Lyase from Erwinia carotovora subsp. atroseptica

Plasmids pKK223.KA-AT, pKK223.KA, pKK223.AT and pKK223-3 weretransformed into the E. coli MG1655 strain. The transformants wererestreaked onto a MOPS minimal media plate containing 1% glucose, 0.5%aminopropanol as a sole nitrogen source, 1 mM IPTG and 100 μg/mLampicillin. Expression of KA-AT, KA and AT genes were induced by theIPTG. A control plate had no IPTG included. The plates were incubated at37° C. for 7 days. On the plate with IPTG, only the strainMG1655/pKK223.KA-AT grew, while all the other three strains did notgrow. On the plate without added IPTG, the strain MG1655/pKK223.KA-ATgrew, but the colonies were significantly smaller than those on theIPTG-containing plate, which corresponds to the lower expression levelsof KA and AT in the uninduced cells. None of the other three strainsgrew on this plate. This indicates that the co-expression of theputative Erwinia KA and AT genes provided sufficient enzyme activitiesthat allowed the E. coli strain MG1655/pKK223.KA-AT to utilizeaminopropanol as a sole nitrogen source. Expression of each individualenzyme of either KA or AT was not sufficient to provide such enzymeactivity in vivo.

Example 19 In vitro Activity of Erwinia Putative Amino Alcohol Kinaseand Amino Alcohol O-Phosphate Lyase

Subcloning of the Erwinia KA-AT Operon into the pBAD.HisB Vector andInduction of Protein Expression

The protein expression levels of Erwinia putative KA and AT enzymesexpressed in MG1655 cells from the pKK223.KA-AT vector were analyzed bySDS-PAGE analysis. The expression level of the Erwinia AT enzyme wasrelatively low, with a new protein band detected at the correctmolecular weight of 46 kD in the soluble fraction of a cell extract,while no new protein band was detected at the size predicted for the KAenzyme.

In an effort to improve the expression of the Erwinia putative KA and ATgenes, the KA-AT operon was subcloned into the EcoRI and HindIII sitesof vector pBAD.HisB-EcoRI. pBAD.HisB-EcoRI was derived from thepBAD.HisB vector (Invitrogen), by replacing the NcoI site in pBAD.HisBwith an EcoRI site via QuickChange site-directed mutagenesis(Stratagene, La Jolla, Calif.) using primers OT909 (SEQ ID.#131) & OT910(SEQ ID.#132). In the constructed plasmid pBAD.KA-AT, the KA-AT operonwas placed directly under control of the araB promoter (withoutHis-tag).

The PBAD.KA-AT plasmid was transformed into the E. coli TOP10 strain. A50 mL culture of TOP10/pBAD.KA-AT strain was grown to mid log phase(OD₆₀₀=0.6) in LB, 100 μg/mL ampicillin media at 37° C. with shaking at250 rpm. The culture was induced by addition of L-arabinose to a finalconcentration of 0.1% (w/v), and it was further incubated at 37° C. for5 h before harvesting by centrifugation. The cell pellet was resuspendedin ice cold 50 mM Tris-HCl, pH 8.0, and disrupted by sonication on icewith a Fischer Sonic Model 300 Dismembrator (Fischer, Pittsburgh, Pa.)at 50% power, repeating four cycles of 30 seconds sonication with 60seconds rest in-between each cycle. Each sonicated sample wascentrifuged (15,000×g, 4 min, 4° C.). Clarified cell free extracts wereanalyzed for protein expression level and amino alcohol O-phosphatelyase activity.

Chemical Synthesis of Aminobutanol O-Phosphate and AminopropanolO-Phosphate

The substrate (R,R)-3-amino-2-butanol O-phosphate was synthesized by amethod based on that reported by Ferrari and Ferrari (U.S. Pat. No.2,730,542 [1956]) for phosphoethanolamine: 10 mmol of H₃PO₄ in a 50%(w/v) aqueous solution was mixed with a 50% (w/v) solution of3-amino-2-butanol (˜20:1 (R,R):(S,S) isomers; Bridge Organics;Vicksburg, Mich.) while stirring on ice. After mixing, the solution wasslowly warmed to room temperature and then stirred under vacuum andheated to 70° C. After 1 h at 70° C., the temperature was slowlyincreased to 185° C. and maintained there for an additional 2 h. At thattime, the reaction was cooled to room temperature and the vacuumreleased. The remaining material was dissolved in water, and analysis byNMR indicated that 80% of the starting material was converted to productwith 20% remaining unreacted. No additional products were observed.

The additional substrates (2R,3S)-3-amino-2-butanol O-phosphate and(2S,3R)-3-amino-2-butanol O-phosphate were synthesized by the sameprocedure using a 1:1 mixture of (2R,3S)-3-amino-2-butanol and(2S,3R)-3-amino-2-butanol (synthesized as described in Example 17) asthe starting material. DL-1-amino-2-propanol O-phosphate,(S)-2-amino-1-propanol O-phosphate, and (R)-2-amino-1-propanolO-phosphate were synthesized by the same procedure usingDL-1-amino-2-propanol, (R)-2-amino-1-propanol, or (S)-2-amino-1-propanolas the starting material.

Analysis of the Aminopropanol O-Phosphate Lyase Activity Encoded by thePutative Erwinia KA-AT Operon

The aminopropanol O-phosphate lyase assay was performed as described byJones et al. (1973, Biochem. J. 134:167-182) and G. Gori et al. (1995,Chromatographia 40:336) The formation of propionaldehyde fromaminopropanol O-phosphate was assayed calorimetrically with MBTH, whichallows the detection of aldehyde formation. The reaction was performedas follows. In a 1 mL reaction, 100 μg cell free extract of E. coliTOP10/pBAD.KA-AT was added to 10 mM DL-1-amino-2-propanol O-phosphate in100 mM Tris-HCl, pH 7.8, with 0.1 mM PLP. The reaction was incubated at37° C. for 10 min and 30 min, with an aliquot of 100 μL reaction mixtureremoved at each time point and mixed with 100 μL of 6 mg/mL MBTH in 375mM glycine-HCl, pH 2.7. This mixture was incubated at 100° C. for 3 min,cooled on ice for 15-30 s, and 1 mL of 3.3 mg/mL FeCl₃.6H₂O (in 10 mMHCl) was added, followed by incubation for 30 min at room temperature.The absorbance of the reaction mixture which contains the aldehyde-MBTHadduct, was measured at 670 nm. The results of the assay are listed inTable 17. In the presence of the aminopropanol phosphate substrate, PLPand cell free extract, formation of aldehyde was detected, as indicatedby an Abs₆₇₀ that was higher than the control background of up to 0.3.In the absence of either the substrate or the cell free extract, noaldehyde formation was detected. In the absence of added PLP, somewhatless amount aldehyde was detected, presumably due to the presence of PLPin the cell free extract. Cell free extract of the uninducedTOP10/pBAD.KA-AT-culture did not produce any detectable aldehyde in thereaction. These results indicated that the putative Erwinia aminoalcohol O-phosphate lyase does catalyze the conversion of aminopropanolO-phosphate to propionaldehyde.

TABLE 17 Aminopropanol O-phosphate lyase assay. Sample 1 was the cellfree extract of a non-induced control of E. coli TOP10/pBAD.KA-AT.Samples 2-5 contained the cell free extract of the induced culture E.coli TOP10/pBAD.KA-AT. Enzyme Induction Aminopropanol extract Sample by0.1% O- (100 OD₆₇₀, OD₆₇₀, Number arabinose phosphate PLP μg/mL) 10 min30 min 1 uninduced (+) (+) (+) 0.262 0.255 2 induced (+) (+) (+) 1.2292.264 3 induced (−) (+) (+) 0.303 0.223 4 induced (+) (−) (+) 0.8551.454 5 induced (+) (+) (−) 0.156 0.065Analysis of the Activity of the Erwinia Amino Alcohol O-Phosphate LyaseTowards Aminobutanol O-Phosphate Substrate

The activity of the amino alcohol O-phosphate lyase towards theaminobutanol O-phosphate substrates was studied under the sameconditions as described above. The reaction was carried out at 37° C.overnight in a 1 mL reaction that contained 100 μg of cell free extractof E. coli TOP10/pBAD.KA-AT, 10 mM aminobutanol O-phosphate (either themixture of (R,R)+(S,S) or the mixture of (R,S)+(S,R) isomers describedin Example 19) in 100 mM Tris-HCl, pH 7.8, with 0.1 mM PLP. An aliquotof 100 μL reaction mixture was removed and the 2-butanone product wasdetected using the MBTH derivatization method described in the GeneralMethods. The two peaks representing the derivatized 2-butanone isomerswere observed. Therefore the Erwinia amino alcohol O-phosphate lyase isan aminobutanol phosphate phospho-lyase in addition to an aminopropanolphosphate phospho-lyase.

Analysis of the Activity of the Erwinia Amino Alcohol O-Phosphate LyaseTowards Stereoisomers of Aminopropanol O-Phosphate and AminobutanolO-Phosphate

The activity of the Erwinia amino alcohol O-phosphate lyase towardsvarious stereoisomers of aminopropanol O-phosphate and aminobutanolO-phosphate was studied under the same conditions as described above. Inthe presence of the Erwinia amino alcohol O-phosphate lyase, both (R)and (S)-2-amino-1-propanol O-phosphate were converted to propanone bythe enzyme, but the product yield was much higher with the (S) isomer.The enzyme also produced butanone from both mixtures of3-amino-2-butanol O-phosphate isomers, with a higher product yield foundin the reaction containing the (R,S) and (S,R) substrate isomers. Bothpropanone and butanone products were derivatized by MBTH, and detectedby HPLC as described in General Methods.

Optimization of the Gene Expression Level for the Erwinia Amino AlcoholKinase and Amino Alcohol O-Phosphate Lyase

In order to improve the expression levels for the Erwinia amino alcoholkinase and the amino alcohol O-phosphate lyase in E. coli, codonoptimized coding regions for both enzymes (named EKA: SEQ ID NO:155 andEAT: SEQ ID NO:156 respectively) were synthesized by DNA2.0 (RedwoodCity, Calif.). Each coding region was synthesized with 5′ and 3′ tailsincluding restriction sites for cloning: EKA has 5′ BbsI and 3′ EcoRI,HindIII sites; EAT has 5′ EcoRI and 3′ HindIII sites. The EKA and EATcoding regions were provided from DNA2.0 as plasmids pEKA and pEAT,which were in the pJ51 vector of DNA2.0. The EKA optimized coding regionwas subcloned by ligating a BbsI and HindIII digested fragment of pEKAinto the pBAD.HisB vector between the NcoI and HindIII sites, togenerate plasmid PBAD.EKA. In the resulting plasmid the coding region is5′ to the His tag, so a coding region for an N-terminus His₆ tag fusedto the Erwinia amino alcohol kinase was constructed by performing aQuickChange site-directed mutagenesis reaction using primers SEQ IDNO:157 and SEQ ID NO:158 to generate vector pBAD.His-EKA.

pBAD.His-EKA was transformed into E. coli strain BL21-Al (F⁻ ompt hsdSB(rB⁻ mB⁻) gal dcm araB::T7RNAP-tetA; Invitrogen) to produce strainBL21-Al/pBAD.HisA-EKA. A 50 mL culture of BL21-Al/pBAD.HisA-EKA wasgrown to mid-log stage (OD₆₀₀=0.6), induced with 0.1% arabinose, andfurther incubated at 30° C. overnight. Cell free extracts were preparedby sonication. The His₆-tagged fusion protein of Erwinia amino alcoholkinase was purified using the ProBond™ Purification System (Invitrogen)under non-denaturing purification conditions following themanufacturer's instructions.

The kinase activity of the His₆-tagged Erwinia amino alcohol kinase isanalyzed by the ADP Quest Assay (DiscoveRx, Fremont, Calif.) followingthe manufacturer's instructions. This is a biochemical assay thatmeasures the accumulation of ADP, a product of the amino alcohol kinasereaction using either aminopropanol or aminobutanol as substrate. 10 mMsubstrate is mixed with His₆-tagged Erwinia amino alcohol kinase, in 100mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 2 mM KCl, 0.1 mM ATP, and incubated at37° C. for 1 h in a 0.2 mL reaction. ADP reagent A (100 μL) and ADPreagent B (200 μL) are added and the mixture is incubated at roomtemperature for 30 min. The fluorescence signal indicating activity ismeasured with excitation wavelength of 530 nm and emission wavelength of590 nm.

Example 20 Expression of Entire Pathway 3 Construction of VectorpCLBudAB-ter-T5chnA

The vector pTrc99a::BudABC (described in Example 13) is digested withEcoRI, and the DNA is treated with Klenow DNA polymerase to blunt theends. The blunted vector is subsequently digested with SpeI to yield a2.5 kb fragment containing the budA and budB genes. The vectorpCL1925-ter-T5chnA (described in Example 13) is digested with HindIII,and the DNA was treated with Klenow DNA polymerase to blunt the ends.The blunted vector is subsequently digested with XbaI to yield a 4.6 kbfragment which is then ligated to the budAB fragment frompTrc99a::BudABC. The resulting plasmid, designated pCLBudAB-ter-T5chnA,is used to transform E. coli Top10 cells, and single colonies arescreened for proper plasmid structure by PCR using primers pCL1925vecF(SEQ ID NO:62) and N84seqR3 (SEQ ID NO:159). Plasmid is prepared from asingle colony which yields a PCR product of the expected size of 1.4 kb.

Construction of Vector pKK223.KA-AT-APT

The APT gene is amplified from the vector PBAD.APT (described in Example16) by PCR using primers APTfor (SEQ ID NO:162; 5′ includes RBS and SmaIsite) and APTrev (SEQ ID NO:163; 3′ adds SmaI site). The product ofexpected size of 1.7 kbp is gel purified and digested with SmaI to yieldblunt ends. The vector pKK223.KA-AT (described in Example 18) isdigested with PstI, and the DNA is treated with Klenow DNA polymerase toblunt the ends. The resulting DNA fragment is ligated with theSmaI-digested PCR product, and the ligation product is used to transformE. coli Top10 cells. Individual ampicillin resistant colonies arescreened by PCR using primers OT872 (SEQ ID NO:127) and APTrev (SEQ IDNO:163). The presence of a PCR product of the expected size of 4.1 kbpindicates that the gene encoding APT is present and oriented in the samedirection as the genes encoding KA and AT. The sequence of the insert isverified using the primers APTseqRev (SEQ ID NO:160) and APTseqFor (SEQID NO:161). This plasmid is named pKK223.KA-AT-APT. Proper expression ofall three genes is verified by growing a 5 mL culture ofTop10/pKK223.KA-AT-APT in LB+100 μg/mL ampicillin at 37° C. withshaking. When the OD₆₀₀ reaches ˜0.8, expression of the genes on theplasmid is induced by addition of IPTG to 0.4 mM. The expression isevaluated by SDS PAGE and activity assays as described above.

Construction of 2-butanol Production Strain and Production of 2-butanoneand 2-butanol

E. coli strain MG1655 is transformed with both pKK223.KA-AT-APT andpCLBudAB-ter-T5chnA, and transformants selected for ampicillin andspectinomycin resistance, indicative of the presence of the plasmids.The cells are inoculated into shake flasks (approximately 175 mL totalvolume) containing 50 or 150 mL of TM3a/glucose medium (with appropriateantibiotics) to represent medium and low oxygen conditions,respectively. IPTG is added to 0.4 mM to induce expression of genes frompKK223.KA-AT-APT. As a negative control, MG1655 cells are grown in thesame medium lacking antibiotics. The flasks are inoculated at a startingOD₆₀₀ of ≦0.01 and incubated at 34° C. with shaking at 300 rpm for 24 h.The flasks containing 50 mL of medium are capped with vented caps; theflasks containing 150 mL are capped with non-vented caps to minimize airexchange. The MG1655/pKK223.KA-AT-APT/pCLBudAB-ter-T5chnA straincomprising a 2-butanol biosynthetic pathway produces both 2-butanone and2-butanol under low and medium oxygen conditions while the negativecontrol strain does not produce detectable levels of either 2-butanoneor 2-butanol.

Example 21 Characterization of Glycerol Dehydratase ButanediolDehydratase Activity

Glycerol dehydratase (E.C. 4.2.1.30) and diol dehydratase (E.C.4.2.1.28), while structurally related, are often distinguished in theart based on various differences that include substrate specificity.This example demonstrates that glycerol dehydratase convertsmeso-2,3-butanediol to 2-butanone. The recombinant E. coli strainKLP23/pSYCO12, comprising Klebsiella pneumoniae genes encoding themultiple subunits of glycerol dehydratase (alpha: SEQ ID NO:145 (codingregion) and 146 (protein); beta: SEQ ID NO: 147 (coding region) and 148(protein); and gamma: SEQ ID NO: 149 (coding region) and 150 (protein))and Klebsiella pneumoniae genes encoding the multiple subunits ofglycerol dehydratase reactivase (large subunit, SEQ ID NO: 151 (codingregion) and 152 (protein); and small subunit, SEQ ID NO: 153 (codingregion) and 154 (protein)), is described in Emptage et al. U.S. Pat. No.6,514,733 and in WO 2003089621, which are herein incorporated byreference. A crude, cell free extract of KLP23/pSYCO12 was prepared bymethods known to one skilled in the art. Enzyme assay was performed inthe absence of light in 80 mM HEPES buffer, pH 8.2 at 37° C. with 12 μMcoenzyme B₁₂ and 10 mM meso-2,3-butanediol. The formation of 2-butanonewas monitored by HPLC (Shodex SH-1011 column and SH-G guard column withrefractive index detection; 0.01 M H₂SO₄ as the mobile phase at a flowrate of 0.5 mL/min and a column temperature of 50° C.; 2-butanoneretention time=40.2 min). The rate of 2-butanone formation by theglycerol dehydratase preparation was determined to be 0.4 nmol/min/mg ofcrude protein.

Example 22 Increased Tolerance of Saccharomyces cerevisiae to 2-butanolat Decreased Growth Temperatures

Tolerance levels were determined for yeast strain Saccharomycescerevisiae BY4741 (ATCC 201388) at 25° C. and 30° C. as follows. Thestrain was cultured in YPD medium. Overnight cultures in the absence ofany test compound were started in 25 mL of YPD medium in 150 mL flaskswith incubation at 30° C. or at 25° C. in shaking water baths. The nextmorning, each overnight culture was diluted into a 500 mL flaskcontaining 300 mL of fresh medium to an initial OD₆₀₀ of about 0.1. Theflasks were incubated in shaking water baths at 30° C. or 25° C., usingthe same temperature as used for each overnight culture. The largecultures were incubated for 3 hours and then were split into flasks inthe absence (control) and in the presence of 2.5% or 3.5% of 2-butanol.Growth was followed by measuring OD₆₀₀ for six hours after addition ofthe 2-butanol. The ΔOD₆₀₀ was calculated by subtracting the initialOD₆₀₀ from the final OD₆₀₀ at 6 hours. The percent growth inhibitionrelative to the control culture was calculated as follows: % GrowthInhibition=100−[100(Sample ΔOD₆₀₀/Control ΔOD₆₀₀)]. The results aresummarized in Table 18 below and indicate that growth of strain BY4741was less inhibited by 2.5% and 3.5% 2-butanol at 25° C. than by 2.5% and3.5% 2-butanol at 30° C.

TABLE 18 Growth of Saccharomyces cerevisiae Strain BY4741 at 25° C. and30° C. with 2-Butanol. % 2- Temperature % Growth Butanol ° C. Inhibition2.5 30 95 2.5 25 89 3.5 30 99 3.5 25 97

What is claimed is:
 1. A method for the production of 2-butanolcomprising: a) providing a recombinant microbial production host whichproduces 2-butanol, wherein the recombinant microbial production hostcomprises heterologous DNA molecules encoding polypeptides that catalyzeeach of the following substrate to product conversions: i) pyruvate toalpha-acetolactate, ii) alpha-acetolactate to acetoin, iii) acetoin to2,3-butanediol, iv) 2,3-butanediol to 2-butanone, and v) 2-butanone to2-butanol; b) seeding the production host of (a) into a fermentationmedium comprising a fermentable carbon substrate to create afermentation culture; c) growing the production host in the fermentationculture at a first temperature for a first period of time; d) loweringthe temperature of the fermentation culture to a second temperature; ande) incubating the production host at the second temperature of step (d)for a second period of time; whereby 2-butanol is produced.
 2. Themethod according to claim 1, wherein the fermentable carbon substrate isderived from a grain or sugar source selected from the group consistingof wheat, corn, barley, oats, rye, sugar cane, sugar beets, cassaya,sweet sorghum, and mixtures thereof.
 3. The method according to claim 1,wherein the fermentable carbon substrate is derived from cellulosic orlignocellulosic biomass selected from the group consisting of corn cobs,crop residues, corn husks, corn stover, grasses, wheat straw, barleystraw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse,sorghum, soy, components obtained from milling of grains, trees,branches, roots, leaves, wood chips, sawdust, shrubs and bushes,vegetables, fruits, flowers, animal manure, and mixtures thereof.
 4. Themethod according to claim 1, wherein the fermentable carbon substrate isselected from the group consisting of monosaccharides, oligosaccharides,and polysaccharides.
 5. The method according to claim 1, wherein thefermentation culture is maintained under conditions selected from thegroup consisting of anaerobic conditions and microaerobic conditions. 6.The method according to claim 1, wherein while growing the productionhost in (c) at a first temperature over a first period of time, ametabolic parameter of the fermentation culture is monitored.
 7. Themethod according to claim 6, wherein the metabolic parameter that ismonitored is selected from the group consisting of optical density, pH,respiratory quotient, fermentable carbon substrate utilization, CO₂production, and 2-butanol production.
 8. The method according to claim1, wherein lowering the temperature of the fermentation culture of step(d) occurs at a predetermined time.
 9. The method according to claim 1,wherein the lowering of the temperature of the fermentation culture ofstep (d) coincides with a change in a metabolic parameter.
 10. Themethod according to claim 9, wherein the change in metabolic parameteris a decrease in the rate of 2-butanol production.
 11. The methodaccording to claim 1, wherein the first temperature is from about 25° C.to about 40° C.
 12. The method according to claim 1, wherein the secondtemperature is from about 3° C. to about 25° C. lower than the firsttemperature.
 13. The method according to claim 1, wherein steps (d) and(e) are repeated one or more times.
 14. The method according to claim 1,wherein the recombinant microbial production host is selected from thegroup consisting of Clostridium, Zymomonas, Escherichia, Salmonella,Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Saccharomyces, and Pichia.
 15. A method for theproduction of 2-butanol comprising, a) providing a recombinant microbialproduction host which produces 2-butanol, wherein the recombinantmicrobial production host comprises heterologous DNA molecules encodinga polypeptides that catalyze each of the following substrate to productconversions: i) pyruvate to alpha-acetolactate, ii) alpha-acetolactateto acetoin, iii) acetoin to 3-amino-2-butanol, iv) 3-amino-2-butanol to3-amino-2-butanol phosphate, v) 3-amino-2-butanol phosphate to2-butanone, and vi) 2-butanone to 2-butanol; b) seeding the productionhost of (a) into a fermentation medium comprising a fermentable carbonsubstrate to create a fermentation culture; c) growing the productionhost in the fermentation culture at a first temperature for a firstperiod of time; d) lowering the temperature of the fermentation cultureto a second temperature; and e) incubating the production host at thesecond temperature of step (d) for a second period of time; whereby2-butanol is produced.
 16. The method according to claim 15, wherein thepolypeptide that catalyzes a substrate to product conversion of pyruvateto alpha-acetolactate is acetolactate synthase.
 17. The method accordingto claim 15, wherein the polypeptide that catalyzes a substrate toproduct conversion of alpha-acetolactate to acetoin is acetolactatedecarboxylase.
 18. The method according to claim 15, wherein thepolypeptide that catalyzes a substrate to product conversion of acetointo 3-amino-2-butanol is acetoin aminase.
 19. The method according toclaim 15, wherein the polypeptide that catalyzes a substrate to productconversion of 3-amino-2-butanol to 3-amino-2-butanol phosphate isaminobutanol kinase.
 20. The method according to claim 15, wherein thepolypeptide that catalyzes a substrate to product conversion of3-amino-2-butanol phosphate to 2-butanone is aminobutanol phosphatephospho-lyase.
 21. The method according to claim 15, wherein thepolypeptide that catalyzes a substrate to product conversion of2-butanone to 2-butanol is butanol dehydrogenase.
 22. The methodaccording to claim 16, wherein the acetolactate synthase has an aminoacid sequence having at least 95% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:4, SEQ ID NO:77, and SEQID NO:79 based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix.
 23. The method according to claim 17,wherein the acetolactate decarboxylase has an amino acid sequence havingat least 95% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO: 81, and SEQ ID NO:83 based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix.
 24. The method according to claim 18, wherein the acetoinaminase has an amino acid sequence having at least 95% identity to anamino acid sequence as set forth in SEQ ID NO:122 based on the Clustal Wmethod of alignment using the default parameters of GAP PENALTY=10, GAPLENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 25.The method according to claim 19, wherein the aminobutanol kinase has anamino acid sequence having at least 95% identity to an amino acidsequence as set forth in SEQ ID NO:124 based on the Clustal W method ofalignment using the default parameters of GAP PENALTY=10, GAP LENGTHPENALTY=0.1, and Gonnet 250 series of protein weight matrix.
 26. Themethod according to claim 20, wherein the aminobutanol phosphatephospho-lyase has an amino acid sequence having at least 95% identity toan amino acid sequence as set forth in SEQ ID NO:126 based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix.
 27. The method according to claim 21, wherein the butanoldehydrogenase has an amino acid sequence having at least 95% identity toan amino acid sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:72, SEQ ID NO:75, and SEQ ID NO:91 based on the ClustalW method of alignment using the default parameters of GAP PENALTY=10,GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.28. The method according to claim 1, wherein the polypeptide thatcatalyzes a substrate to product conversion of pyruvate toalpha-acetolactate is acetolactate synthase.
 29. The method according toclaim 1, wherein the polypeptide that catalyzes a substrate to productconversion of alpha-acetolactate to acetoin is acetolactatedecarboxylase.
 30. The method according to claim 1, wherein thepolypeptide that catalyzes a substrate to product conversion of acetointo 2,3-butanediol is butanediol dehydrogenase.
 31. The method accordingto claim 1, wherein the polypeptide that catalyzes a substrate toproduct conversion of 2,3-butanediol to 2-butanone is diol dehydrataseor glycerol dehydratase.
 32. The method according to claim 1, whereinthe polypeptide that catalyzes a substrate to product conversion of2-butanone to 2-butanol is butanol dehydrogenase.
 33. The methodaccording to claim 28, wherein the acetolactate synthase has an aminoacid sequence having at least 95% identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:4, SEQ ID NO:77, and SEQID NO:79 based on the Clustal W method of alignment using the defaultparameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250series of protein weight matrix.
 34. The method according to claim 29,wherein the acetolactate decarboxylase has an amino acid sequence havingat least 95% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO: 81, and SEQ ID NO:83 based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix.
 35. The method according to claim 30, wherein thebutanediol dehydrogenase has an amino acid sequence having at least 95%identity to an amino acid sequence selected from the group consisting ofSEQ ID NO:6, SEQ ID NO:85, SEQ ID NO:87, and SEQ ID NO:89 based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix.
 36. The method according to claim 31, wherein the dioldehydratase or glycerol dehydratase comprises fused large, medium andsmall subunits and has at least 95% identity to an amino acid sequencecomprising all three of the amino acid sequences encoding large, mediumand small subunits, selected from the group consisting of: a) SEQ IDNO:8, SEQ ID NO:10, and SEQ ID NO:12; b) SEQ ID NO:93, SEQ ID NO:95, andSEQ ID NO:97; c) SEQ ID NO:99, SEQ ID NO:101, and SEQ ID NO:103; d) SEQID NO:105, SEQ ID NO:107, and SEQ ID NO:109; e) SEQ ID NO:135, SEQ IDNO:136, and SEQ ID NO:137; f) SEQ ID NO:138, SEQ ID NO:139, and SEQ IDNO:140; g) SEQ ID NO:146, SEQ ID NO:148, and SEQ ID NO:150; h) SEQ IDNO:141, SEQ ID NO:142, and SEQ ID NO:143; and i) SEQ ID NO:164, SEQ IDNO:165, and SEQ ID NO:166; based on the Clustal W method of alignmentusing the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1,and Gonnet 250 series of protein weight matrix.
 37. The method accordingto claim 32, wherein the butanol dehydrogenase has an amino acidsequence having at least 95% identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:14, SEQ ID NO:72, SEQ ID NO:75,and SEQ ID NO:91 based on the Clustal W method of alignment using thedefault parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet250 series of protein weight matrix.
 38. The method according to claim15, wherein the fermentable carbon substrate is derived from a grain orsugar source selected from the group consisting of wheat, corn, barley,oats, rye, sugar cane, sugar beets, cassaya, sweet sorghum, and mixturesthereof.
 39. The method according to claim 15, wherein the fermentablecarbon substrate is derived from cellulosic or lignocellulosic biomassselected from the group consisting of corn cobs, crop residues, cornhusks, corn stover, grasses, wheat straw, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sorghum, soy, componentsobtained from milling of grains, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animalmanure, and mixtures thereof.
 40. The method according to claim 15,wherein the fermentable carbon substrate is selected from the groupconsisting of monosaccharides, oligosaccharides, and polysaccharides.41. The method according to claim 15, wherein the fermentation cultureis maintained under conditions selected from the group consisting ofanaerobic conditions and microaerobic conditions.
 42. The methodaccording to claim 15, wherein while growing the production host in (c)at a first temperature over a first period of time, a metabolicparameter of the fermentation culture is monitored.
 43. The methodaccording to claim 42, wherein the metabolic parameter that is monitoredis selected from the group consisting of optical density, pH,respiratory quotient, fermentable carbon substrate utilization, CO₂production, and 2-butanol production.
 44. The method according to claim15, wherein lowering the temperature of the fermentation culture of step(d) occurs at a predetermined time.
 45. The method according to claim15, wherein the lowering of the temperature of the fermentation cultureof step (d) coincides with a change in a metabolic parameter.
 46. Themethod according to claim 45, wherein the change in metabolic parameteris a decrease in the rate of 2-butanol production.
 47. The methodaccording to claim 15, wherein the first temperature is from about 25°C. to about 40° C.
 48. The method according to claim 15, wherein thesecond temperature is from about 3° C. to about 25° C. lower than thefirst temperature.
 49. The method according to claim 15, wherein steps(d) and (e) are repeated one or more times.
 50. The method according toclaim 15, wherein the recombinant microbial production host is selectedfrom the group consisting of Clostridium, Zymomonas, Escherichia,Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,Corynebacterium, Brevibacterium, Saccharomyces, and Pichia.